Aniline-based wdr5 protein-protein interaction inhibitor, and preparation method and use thereof

ABSTRACT

The present disclosure discloses a WDR5 protein-protein interaction inhibitor, including a compound having a structure represented by general formula (I). Experiments show that the inhibitor acts on a WDR5 protein and an interacting protein thereof including, but not limited to, MLL, selectively inhibits the proliferation of leukemia cells, and inhibits the methylation of H3K4 and the expression of downstream Hox/Meis-1 gene at the cellular level. The present disclosure also discloses a method for preparing the inhibitor and use thereof in the preparation of a drug for treating acute leukemia and other related diseases.

TECHNICAL FIELD

The present disclosure relates to an aniline compound in medicinalchemistry, a preparation method thereof, and use thereof. As a WDR5protein-protein interaction inhibitor, the compound inhibits the levelof a histone methyltransferase and the expression of a downstreamhematopoietic gene at the cellular level, inhibits the growth oftransplanted tumors in nude mice at the animal level, and can be used totreat diseases such as acute leukemia.

BACKGROUND

A WDR5 protein includes 334 amino acids, and contains 7 WD40 repeatdomains. Structural studies have shown that the WD40 repeat domains forma 7-blade propeller structure, each blade including 4 antiparallelchains. Such a structural feature enables WDR5 to interact with manyother proteins, for example, WDR5-MLL1, WDR5-MYC, and WDR5-MOF/HAT. WDR5may also bind to H3K4me2. Studies have shown that the WDR5 may stabilizeor assemble a VISA (virus-induced signaling adaptor)-related complex,and plays an important role in type I interferon and antiviral innateimmune response. The WDR5 protein is also highly expressed in bladdercancer, and promotes the proliferation, self-renewal and chemotherapy ofbladder cancer cells by activating the expression of H3K4me3. As animportant cofactor, the WDR5 participates in N-Myc-regulatedtranscriptional activation and tumorigenesis. Inhibition of the WDR5protein is a novel target for the treatment of MYCN-amplifiedneuroblastoma.

The histone methylation plays a key role in many bioprocesses and is animportant research content in the field of epigenetic regulation. MLL1is a histone H3 lysine 4 (H3K4) methyltransferase. MLL1 gene istranslocated and rearranged to form a fusion gene that expresses anoncogenic MLL fusion protein to induce mixed lineage leukemia (MLL1,acute myeloid leukemia and acute lymphatic leukemia). Conventionalchemotherapy for the treatment of leukemia caused by MLL abnormality isundesirable with poor prognosis. No targeted drug is available atpresent. Therefore, it is an urgent need of development of a noveltherapeutic agent for the biological properties of the disease.

The chromosomal translocation of MLL1 only occurs on a monoallelic gene,and there is also a wild-type MLL1. The wild-type MLL1 and a fusionprotein thereof are jointly involved in the occurrence and developmentof mixed lineage leukemia, where enzymatic activity of the wild-typeMLL1 is crucial for the induction of leukemia by the MLL1 fusionprotein. Therefore, specifically inhibiting the enzymatic activity ofthe wild-type MLL1 can achieve the effect of leukemia treatment.

MLL1 present alone has very poor catalytic activity, and can onlycatalyze the monomethylation of H3K4. When MLL1 forms a core complexwith WDR5, RbBP5, Ash2L, and DPY30, the enzymatic catalysis activity isgreatly enhanced, especially the catalytic activity for H3K4me2. As abridge linking a C-terminal WIN motif of MLL1 to other proteins, theWDR5 plays a vital role in the formation of the complex. Therefore,interfering with the WDR5 protein-protein interaction using amicromolecular inhibitor is an effective way to inhibit the enzymaticcatalysis activity of MLL1, thereby inhibiting the expression ofdownstream Hox and Meis-1 genes, and thus blocking the progression ofleukemia.

SUMMARY OF THE INVENTION

Object of the Disclosure: An object of the present disclosure is toprovide a WDR5 protein-protein interaction inhibitor, which inhibits thelevel of a histone methyltransferase and the expression of a downstreamhematopoietic gene at the cellular level and inhibits the growth oftransplanted tumors in nude mice at the animal level; and use thereof inthe treatment of a drug for an indication related to a MLL enzymefunction. The inhibitor can be used to treat diseases such as acuteleukemia.

Technical Solution: The present disclosure discloses a micromolecularcompound that may interfere with WDR5 protein-protein interaction,inhibits the catalytic activity of a MLL1 for methyl transfer anddown-regulates the expression levels of Hox and Meis-1 genes byinterfering with the WDR5 protein-protein interaction, thereby inducingthe apoptosis of leukemia cells for use in the treatment of acuteleukemia. The compound of the present disclosure has a structure below:

where X represents CH or N;

Y represents C or N;

R¹, R² and R³ are identical to or different from each other, and areeach independently selected from the group consisting of hydrogen, C₁-C₆alkyl, C₃-C₆ cycloalkyl, C₁-C₆ alkyl substituted with C₁-C₆ alkoxy,nitro, halo, cyano, aldehyde group, hydroxyl, —NR⁷R⁸, or

formed by linking R² and R³, where R⁷ and R⁸ are identical to ordifferent from each other, and each independently represent hydrogen orC₁-C₄ alkyl, or 3- to 7-membered nitrogenous heterocycle formed bylinking R⁷ and R⁸;

R⁴ represents morpholinyl, piperazinyl, 4-substituted piperazinyl,4-substituted homopiperazinyl, 3-substituted piperazinyl, or2-substituted piperazinyl, where a substituent is C₁-C₄ alkyl, 3- to7-membered cycloalkyl, hydroxyalkyl, or phenyl;

R⁵ represents nitro, amino, phenyl, substituted phenyl, 5- to 6-memberedoxygenic or nitrogenous aromatic heterocycle, substituted 5- to6-membered oxygenic or nitrogenous aromatic heterocycle, or —NHCOR⁹,where R⁹ represents hydroxyl, C₁-C₆ alkoxy, phenyl, substituted phenyl,5- to 6-membered oxygenic or nitrogenous aromatic heterocycle, orsubstituted 5- to 6-membered oxygenic or nitrogenous aromaticheterocycle, where a substituent is C₁-C₄ alkyl, C₁-C₄ alkoxy, halo,cyano, —NHCOR¹⁰, —CONR¹¹R¹² or —COOR¹⁰, where R¹⁰ represents hydrogen,C₁-C₆ alkyl, C₁-C₆ amino-substituted alkyl, 3- to 7-membered cycloalkyl,3- to 7-membered nitrogenous or oxygenic heterocycle, or phenyl; and R¹¹and R¹² independently represent hydrogen, C₁-C₆ alkyl, phenyl orsubstituted phenyl, substituted or unsubstituted 3- to 7-memberednitrogenous or oxygenic heterocycle, or 3- to 7-membered nitrogenous oroxygenic heterocycle formed by R¹¹ and R¹²; and

R⁶ represents hydrogen, halo, methyl, trifluoromethyl, amino, orsubstituted amino, where a substituent is C₁-C₄ alkyl, or allyl;

R¹, R² and R³ are identical to or different from each other, and areeach independently selected from the group consisting of hydrogen, halo,methyl, nitro, cyano, aldehyde group, methoxyl, —NR⁷R⁸, or

performed by linking R² and R³, where R⁷ and R⁸ are identical to ordifferent from each other, and each independently represent hydrogen, orC₁-C₄ alkyl, or 3- to 5-membered nitrogenous heterocycle formed bylinking R⁷ and R⁸;

R⁴ is preferably morpholinyl, piperazinyl, or 4-substituted piperazinyl,where a substituent is methyl, ethyl, cyclopropyl, hydroxyethyl, orphenyl;

R⁵ is preferably nitro, amino, —NHCOR⁹, furyl, pyrimidinyl, pyridinyl,substituted triazolyl, or substituted phenyl, where a substituent is amono- or di-substituted halo, —NHCOR¹⁰, —CONR¹¹R¹², or —COOR¹⁰, where R⁹represents phenyl; R¹⁰ represents hydrogen, C₁-C₆ alkyl, C₁-C₆amino-substituted alkyl, 3- to 7-membered cycloalkyl, 3- to 7-memberednitrogenous or oxygenic heterocycle, or phenyl; and R¹¹ and R¹²independently represent hydrogen, C₁-C₆ alkyl, phenyl or substitutedphenyl, substituted or unsubstituted 3- to 7-membered nitrogenous oroxygenic heterocycle, or 3- to 7-membered nitrogenous or oxygenicheterocycle formed by linking R¹¹ and R¹².

The present disclosure further includes a pharmaceutically acceptablesalt of Compound (I) and a solvate thereof, which have the samepharmacological effects as Compound (I).

The present disclosure discloses a pharmaceutical composition, whichcomprises Compound (I), or a pharmaceutical salt thereof, or a solvate,and one or more pharmaceutical carriers, diluents and excipients.

The present disclosure further provides use of the compound of formula(I) or a pharmaceutical salt thereof or a solvate in the preparation ofa drug for treating a disease mediated by the enzyme by inhibiting WDR5protein-protein interaction. The disease, for example, MLL gene-fusedleukemia, may be treated by inhibiting the enzyme activity of MLL1.

The clinically used dose of the compound of the present disclosure is0.01 mg to 1000 mg/day, or may deviate from this range according to thedisease severity or different dosage forms.

In some embodiments, the compound of formula (I) may contain an alkalinefunctional group adequate to form a salt. Representative salts include apharmaceutical inorganic acid salt, including a hydrochloride, ahydrobromide, and a sulfate; and a pharmaceutical organic acid salt,preferably including an acetate, a trifluoroacetate, a lactate, asuccinate, a fumarate, a maleate, a citrate, a benzoate, amethanesulfonate, a p-benzoate or a p-toluenesulfonate.

Further, the present disclosure also discloses a method for preparing arelated compound of formula (I), including the following processes:

Beneficial effects: The aniline compound of the present disclosure has astrong WDR5 protein-protein interaction inhibiting activity, may reducethe enzymatic catalysis activity of MLL1, down-regulates the expressionof Hox and Meis-1 genes at the cellular level, and is useful in thetreatment of an indication related to a WDR5 enzyme function. Therelated indication refers to a hematological tumor, including diseasessuch as acute leukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of the title compound of Example 72 on theintracellular enzymatic catalysis activity of MLL1, as detected by aWestern blot experiment;

FIG. 2 shows down-regulation of the title compound of Example 72 on theintracellular expression of Hoxa9 and Meis-1 genes, as detected by aRT-PCR experiment; and

FIG. 3 shows the inhibition level of the title compound of Example 72 ona tumor at the animal level detected using a MV4-11 nude mouse xenograftmodel.

DETAILED DESCRIPTION Example 1

N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)quinazoline-4-amine Step 1:Preparation of 2-(4-methylpiperazin-1-yl)-5-nitrophenylamine

2-fluoro-5-nitrophenylamine (2 g, 12.8 mmol) was dissolved in DMF (20mL), and then DIPEA (3.3 g, 25.6 mmol) and 1-methylpiperazine (25.6mmol) were added. The mixture was stirred at 100° C. for 8 hrs. 100 mLof water was added to the reaction mixture, which was extracted withethyl acetate (3×50 mL). The organic layers were combined, dried overanhydrous Na₂SO₄, and distilled under reduced pressure, to obtain acrude product, which was washed with CH₃CN to obtain a yellow solid.Yield 53.0%; m.p. 200° C.-202° C. ¹H NMR (300 MHz, DMSO-d₆) δ 7.53 (d,J=2.4 Hz, 1H), 7.42 (dd, J=8.8, 2.4 Hz, 1H), 7.04 (d, J=8.7 Hz, 1H),5.26 (s, 2H), 3.11 (s, 4H), 2.58 (s, 4H), 2.24 (s, 3H). HRMS (ESI):calcd. for m/z C₁₁H₁₇N₄O₂, [M+H]⁺ 237.1316, found 237.1342.

Step 2: Preparation of Titled Compound

N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)quinazoline-4-amine2-(4-methylpiperazin-1-yl)-5-nitrophenylamine (0.2 g, 0.846 mmol) wasdissolved in isopropanol (10 mL), and then 1 mL of concentrated HCl and4-chloroquinazoline (0.27 g, 1.69 mmol) were added. The mixture wasrefluxed for 18 hrs. The reaction mixture was distilled under reducedpressure until a solid precipitated, cooled, and filtered under suction.The filter cake was washed with isopropanol (5 mL×3), and dried toobtain a yellow solid. Yield 63.5%; m.p.>250° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 9.57 (s, 1H), 8.61 (s, 2H), 8.43 (s, 1H), 8.07 (s, 1H), 7.86(d, J=10.0 Hz, 2H), 7.69 (s, 1H), 7.30 (s, J=8.8 Hz, 1H), 3.09 (s, 4H),2.32 (s, 4H), 2.13 (s, 3H). HRMS (ESI): calcd. for m/z C₁₉H₂₀N₆O₂,[M+H]⁺ 365.1720, found 365.1722. HPLC (100% MeOH): t_(R)=7.772 min,99.16%.

Example 2

6-chloro-N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4-amine

Following the method in Step 2 of Example 1, 4-chloroquinazoline wasreplaced by 4, 6-dichloropyrimidine (1.69 mmol), to obtain a yellowsolid. Yield 65.8%; m.p. 169° C.-171° C.; ¹H NMR (300 MHz, DMSO-d₆) δ9.36 (s, 1H), 8.48 (s, 1H), 8.42 (d, J=2.7 Hz, 1H), 8.02 (dd, J=8.9, 2.8Hz, 1H), 7.23 (d, J=9.0 Hz, 1H), 6.81 (s, 1H), 3.04 (t, J=4.6 Hz, 4H),2.35 (t, J=4.7 Hz, 4H), 2.16 (s, 3H). HRMS (ESI): calcd. for m/zC₁₅H₁₇ClN₆O₂, [M+H]⁺ 349.1174, found 349.1177. HPLC (100% MeOH):t_(R)=6.097 min, 96.84%.

Example 3

N⁴,N⁴-dimethyl-N⁶-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4,6-diamine

The title compound of Example 2 (0.35 mmol) was dissolved in DMF (5 mL),and then DIPEA (1.5 mmol) and dimethylamine (1 mmol) were added. Themixture was kept at 120° C. for 4 hrs. The reaction mixture was dilutedwith ethyl acetate (20 mL), washed with water (10 mL×3), and then washedwith a saturated sodium chloride solution. The organic layer was driedover anhydrous sodium sulfate, filtered under suction, and spin-dried.The crude product was purified by silica gel column chromatography(dichloromethane:methanol=50:1) to obtain a yellow solid. Yield 49.5%;m.p. 152° C.-154° C.; ¹H NMR (300 MHz, CDCl₃) δ 8.69 (d, J=2.7 Hz, 1H),8.28 (s, 1H), 7.80 (dd, J=8.7, 2.7 Hz, 1H), 7.06 (d, J=8.8 Hz, 1H), 6.94(s, 1H), 5.82 (s, 1H), 3.03 (s, 6H), 2.94 (t, J=4.8 Hz, 4H), 2.65-2.48(m, 4H), 2.30 (s, 3H). HRMS (ESI): calcd. for m/z C₁₇H₂₃N₇O₂, [M+H]⁺358.1986, found 358.1992. HPLC (100% MeOH): t_(R)=4.709 min, 99.20%.

Example 4

6-chloro-2-methyl-N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4-amine

Following the method in Step 2 of Example 1, 4-chloroquinazoline wasreplaced by 4,6-dichloro-2-methylpyrimidine, to obtain a yellow solid.Yield 44.8%; m.p. 196° C.-198° C.; ¹H NMR (300 MHz, CDCl₃) δ 9.09 (d,J=2.6 Hz, 1H), 7.97 (dd, J=8.8, 2.7 Hz, 1H), 7.55 (s, 1H), 7.23 (s, 1H),6.59 (s, 1H), 3.02 (t, J=4.8 Hz, 4H), 2.65-2.61 (m, 7H), 2.40 (s, 3H).HRMS (ESI): calcd. for m/z C₁₆H₁₉ClN₆O₂, [M+H]⁺ 363.1331, found363.1335. HPLC (100% MeOH): t_(R)=8.574 min, 97.99%.

Example 5

6-chloro-N⁴-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-2,4-diamine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by 4,6-dichloro-2-aminopyrimidine, toobtain a yellow solid. Yield 77.6%; m.p. 248° C.-251° C.; ¹H NMR (300MHz, CDCl₃) δ 9.13 (s, 1H), 7.94 (dd, J=8.7, 2.6 Hz, 1H), 7.41 (s, 1H),7.20 (d, J=2.6 Hz, 1H), 5.16 (s, 2H), 3.00 (t, J=4.8 Hz, 4H), 2.64 (s,4H), 2.41 (s, 3H). HRMS (ESI): calcd. for m/z C₁₅H₁₈ClN₇O₂, [M+H]⁺364.1283, found 364.1289. HPLC (100% MeOH): t_(R)=5.850 min, 97.14%.

Example 6

6-chloro-5-methyl-N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4-amine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by 4, 6-dichloro-5-methylpyrimidine, toobtain a yellow solid. Yield 60.5%; m.p. 259° C.-261° C.; ¹H NMR (300MHz, CDCl₃) δ 9.55 (s, 1H), 8.57 (s, 1H), 8.18 (s, 1H), 7.95 (dd, J=8.7,2.7 Hz, 1H), 7.27 (s, 1H), 3.02 (t, J=4.8 Hz, 4H), 2.66 (s, 4H), 2.42(s, 6H). HRMS (ESI): calcd. for m/z C₁₆H₁₉ClN₆O₂, [M+H]⁺ 363.1331, found363.1349. HPLC (100% MeOH): t_(R)=7.673 min, 97.05%.

Example 7

6-chloro-N⁴-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4,5-diamine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by 4,6-dichloro-5-aminopyrimidine, toobtain a yellow solid. Yield 84.7%; m.p. 185° C.-187° C.; ¹H NMR (300MHz, DMSO-d₆) δ 8.65 (d, J=2.6 Hz, 1H), 8.22 (s, 1H), 7.98-7.91 (m, 2H),7.27 (d, J=8.9 Hz, 1H), 5.41 (s, 2H), 2.91 (br s, 4H), 2.47 (br s, 4H),2.20 (s, 3H). HRMS (ESI): calcd. for m/z C₁₅H₁₈ClN₇O₂, [M+H]⁺ 364.1283,found 364.1286. HPLC (100% MeOH): t_(R)=6.581 min, 98.22%.

Example 8

6-chloro-2-methyl-N⁴-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4,5-diamine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by4,6-dichloro-2-methyl-5-aminopyrimidine, to obtain a yellow solid. Yield67.8%; m.p. 248° C.-250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.87 (d, J=2.8Hz, 1H), 8.22 (s, 1H), 7.94 (dd, J=8.8, 2.8 Hz, 1H), 5.12 (s, 2H), 2.98(t, J=4.8 Hz, 4H), 2.46 (s, 4H), 2.32 (s, 3H), 2.21 (s, 3H). HRMS (ESI):calcd. for m/z C₁₆H₂₀ClN₇O₂, [M+H]⁺ 378.1440, found 378.1440. HPLC (100%MeOH): t_(R)=6.251 min, 97.99%.

Example 9

N⁴-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4,5-diamine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by 4-chloro-5-aminopyrimidine, toobtain a yellow solid. Yield 43.7%; m.p.>250° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 8.25 (s, 1H), 7.82 (dd, J=7.4, 1.9 Hz, 1H), 7.81-7.77 (m,2H), 7.67 (s, 1H), 7.04 (d, J=7.5 Hz, 1H), 5.82 (s, 2H), 3.20 (t, J=5.3Hz, 4H), 2.98 (t, J=5.3 Hz, 4H), 2.60 (s, 3H). HRMS (ESI): calcd. form/z C₁₅H₁₉N₇O₂, [M+H]⁺ 330.1673, found 330.1675. HPLC (100% MeOH):t_(R)=7.673 min, 97.05%.

Example 10

N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-2-amine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by 2-bromopyrimidine, to obtain ayellow solid. Yield 57.5%; m.p. 195° C.-197° C.; ¹H NMR (300 MHz, CDCl₃)δ 9.47 (s, 1H), 8.54 (d, J=3.0 Hz, 2H), 8.03 (s, 1H), 7.88 (d, J=3.0 Hz,1H), 7.22 (d, J=9.0 Hz, 1H), 6.85 (t, J=9.0 Hz, 1H), 3.04 (t, J=5.6 Hz,4H), 2.70 (br s, 4H), 2.42 (s, 3H). HRMS (ESI): calcd. for m/zC₁₅H₁₈ClN₇O₂, [M+H]⁺ 315.1564, found 315.1566. HPLC (100% MeOH):t_(R)=16.340 min, 96.09%.

Example 11

N²-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-2,4-diamine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by 2-chloropyrimidine-4-amine, toobtain a yellow solid. Yield 45.7%; m.p. 210° C.-212° C.; ¹H NMR (300MHz, DMSO-d₆) δ 9.30 (s, 1H), 7.94 (d, J=6.0 Hz, 1H), 7.80 (dd, J=8.7Hz, 1H), 7.63 (s, 1H), 7.32 (d, J=8.8 Hz, 1H), 6.81 (br s, 2H), 6.03 (d,J=5.8 Hz, 1H), 2.93 (t, J=4.7 Hz, 4H), 2.54 (br s, 4H), 2.26 (s, 3H).HRMS (ESI): calcd. for m/z C₁₅H₁₉N₇O₂, [M+H]⁺ 330.1673, found 330.1679.HPLC (100% MeOH): t_(R)=6.354 min, 96.85%.

Example 12

N²-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyridin-2,3-diamine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by 2-chloropyridin-3-amine, to obtain ayellow solid. Yield 36.6%; m.p. 210° C.-212° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 9.06 (d, J=2.8 Hz, 1H), 7.77 (dd, J=8.8, 2.8 Hz, 1H),7.68-7.59 (m, 2H), 7.28 (d, J=8.8 Hz, 1H), 7.77 (dd, J=7.6, 1.7 Hz, 1H),6.75 (dd, J=7.6, 1.7 Hz, 1H), 4.94 (s, 2H), 2.90 (t, J=4.8 Hz, 4H), 2.54(s, 4H), 2.25 (s, 3H). HRMS (ESI): calcd. for m/z C₁₅H₂₀N₆O₂, [M+H]⁺329.1720, found 329.1722. HPLC (100% MeOH): t_(R)=4.151 min, 95.29%.

Example 13

6-chloro-N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)-5-nitropyrimidine-4-amine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by 4,6-dichloro-5-nitropyrimidine, toobtain a yellow solid. Yield 53.2%; m.p.>250° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 10.20 (s, 1H), 8.85 (d, J=6.5 Hz, 1H), 8.16 (d, J=6.0 Hz,2H), 7.62 (t, J=9.5 Hz, 1H), 3.52 (br s, 4H), 2.42 (br s, 4H), 2.22 (s,3H). HRMS (ESI): calcd. for m/z C₁₅H₁₆ClN₇O₄, [M+H]⁺ 394.0671, found394.0674. HPLC (100% MeOH): t_(R)=6.083 min, 97.01%.

Example 14

4-chloro-6-((2-(4-methylpiperazin-1-yl)-5-nitrophenyl)amino)pyrimidine-5-carbonitrile

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by4,6-dichloropyrimidine-5-carbonitrile, to obtain a yellow solid. Yield82.7%; m.p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.39 (d, J=2.8 Hz, 1H),8.22 (s, 1H), 8.06 (dt, J=9.0, 3.1 Hz, 1H), 7.32 (d, J=8.9 Hz, 1H), 3.21(br s, 4H), 2.84 (br s, 4H), 2.47 (s, 3H). HRMS (ESI): calcd. for m/zC₁₆H₁₆ClN₇O₂, [M+H]⁺ 374.1133, found 374.1135. HPLC (100% MeOH):t_(R)=5.679 min, 98.83%.

Example 15

4-chloro-6-((2-(4-methylpiperazin-1-yl)-5-nitrophenyl)amino)pyrimidine-5-carbaldehyde

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by4,6-dichloropyrimidine-5-carbaldehyde, to obtain a yellow solid. Yield:75.9%; m.p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 11.93 (s, 1H), 10.05(s, 1H), 9.05 (d, J=2.6 Hz, 1H), 8.36 (d, J=1.6 Hz, 1H), 8.01 (dt,J=8.9, 2.3 Hz, 1H), 7.36 (dd, J=8.9, 1.8 Hz, 1H), 3.04-2.87 (m, 4H),2.56 (t, J=4.6 Hz, 4H), 2.26 (s, 3H). HRMS (ESI): calcd. for m/zC₁₆H₁₇ClN₆O₃, [M+H]⁺ 377.1130, found 377.1132. HPLC (100% MeOH):t_(R)=6.472 min, 98.17%.

Example 16

6-chloro-5-methoxyl-N-(2-(4-methylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4-amine

Following the preparation method in Step 2 of Example 1,4-chloroquinazoline was replaced by4,6-dichloropyrimidine-5-carbaldehyde, to obtain a yellow solid. Yield88.9%; m.p.>250° C.; ¹H NMR (300 MHz, CDCl₃) δ 9.59 (d, J=2.7 Hz, 1H),8.68 (s, 1H), 8.45 (d, J=3.5 Hz, 1H), 7.96 (dd, J=8.8, 2.7 Hz, 1H), 7.26(d, J=2.1 Hz, 1H), 4.06 (s, 1H), 3.06-2.97 (m, 4H), 2.67 (br s, 4H),2.42 (s, 3H). HRMS (ESI): calcd. for m/z C₁₆H₁₉ClN₆O₃, [M+H]⁺ 379.1280,found 379.1286. HPLC (100% MeOH): t_(R)=6.614 min, 95.68%.

Example 17

6-chloro-N⁴-(5-nitro-2-(piperazin-1-yl)phenyl)pyrimidine-4,5-diamineStep 1: Preparation of6-chloro-N⁴-(2-fluoro-5-nitrophenyl)pyrimidine-4,5-diamine

2-fluoro-5-nitrophenylamine (2 g, 12.8 mmol) was dissolved inisopropanol (50 mL), and then 2 mL of concentrated sulfuric acid and4,6-dichloro-5-aminopyrimidine (25.6 mmol) were added. The mixture wasrefluxed for 18 hrs. The reaction mixture was spun until a solidprecipitated, cooled until a solid precipitated, and filtered undersuction. The filter cake was washed with isopropanol (5 mL×3), and driedto obtain a yellow solid. Yield 59.5%; m.p. 209° C.-211° C.; ¹H-NMR (300MHz, DMSO-d₆) δ 9.14 (s, 1H), 8.65-8.62 (m, 1H), 8.06-8.03 (m, 1H), 7.86(s, 1H), 7.58-7.54 (m, 1H), 5.54 (s, 2H). m/z (EI-MS): 284.1 [M]⁺.

Step 2: Preparation of a Target Product:6-chloro-N⁴-(5-nitro-2-(piperazin-1-yl)phenyl)pyrimidine-4,5-diamine

6-chloro-N⁴-(2-fluoro-5-nitrophenyl)pyrimidine-4,5-diamine (0.35 mmol)was dissolved in 5 mL of DMF, and then DIPEA (1.5 mmol) and t-butylpiperazine-1-carboxylate (1.0 mmol) were added. The mixture was kept at50° C. for 5 hrs. The reaction mixture was diluted with ethyl acetate(20 mL), washed with water (10 mL×3), and then washed with a saturatedsodium chloride solution. The organic layer was dried over anhydroussodium sulfate, and spin-dried, to obtain a crude product, which wasdissolved in dichloromethane (20 mL), and then trifluoroacetic acid (2mL) was added dropwise. The mixture was kept at room temperature for 2hrs. The reaction mixture was neutralized with a saturated sodiumbicarbonate solution until no bubbles were generated, extracted withethyl acetate, and spin-dried to obtain a yellow solid, which was washedwith CH₃CN and dried. Yield 65.6%; m.p.>250° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 9.09 (br s, 1H), 8.60 (d, J=2.7 Hz, 1H), 8.33 (s, 1H), 7.89(dd, J=8.9, 2.7 Hz, 1H), 7.83 (s, 1H), 7.23 (d, J=8.9 Hz, 1H), 5.55 (s,2H), 3.21 (s, 4H), 3.42 (t, J=6.0 Hz, 4H). HRMS (ESI): calcd. for m/zC₁₄H₁₇ClN₇O₂, [M+H]⁺ 350.1127, found 350.1131. HPLC (100% MeOH):t_(R)=4.069 min, 96.17%.

Example 18

6-chloro-N⁴-(2-morpholino-5-nitrophenyl)pyrimidine-4,5-diamine

6-chloro-N⁴-(2-fluoro-5-nitrophenyl)pyrimidine-4,5-diamine (0.35 mmol)was dissolved in 5 mL of DMF, and then DIPEA (1.5 mmol) and morpholine(1.0 mmol) were added. The mixture was kept at 50° C. for 5 hrs. Thereaction mixture was diluted with ethyl acetate (20 mL), washed withwater (10 mL×3), and then washed with a saturated sodium chloridesolution. The organic layer was dried over anhydrous sodium sulfate, andspin-dried to obtain a crude product, which was washed with CH₃CN anddried. Yield 71.4%; m.p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.70 (s,1H), 8.32 (s, 1H), 8.08-7.86 (m, 2H), 7.29 (d, J=9.0 Hz, 1H), 5.46 (s,2H), 3.73 (s, 4H), 2.99 (s, 4H). HRMS (ESI): calcd. for m/zC₁₄H₁₆ClN₆O₃, [M+H]⁺ 351.0967, found 351.0967. HPLC (100% MeOH):t_(R)=7.041 min, 98.84%.

Example 19

6-chloro-N⁴-(2-(4-ethylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4,5-diamine

Following the preparation method in Step 2 of Example 18, morpholine wasreplaced by 1-ethylpiperazine to obtain a yellow solid. Yield 46.7%;m.p. 185° C.-187° C.; ¹H NMR (300 MHz, CDCl₃) δ 9.34 (s, 1H), 8.32 (s,1H), 8.18 (s, 1H), 7.93 (dt, J=8.8, 2.7 Hz, 1H), 7.27-7.20 (m, 1H), 3.60(s, 2H), 3.08-2.93 (m, 4H), 2.70 (s, 4H), 2.53 (qd, J=7.2, 2.7 Hz, 2H),1.16 (td, J=7.3, 2.7 Hz, 3H). HRMS (ESI): calcd. for m/z C₁₆H₂₁ClN₇O₂,[M+H]⁺ 378.1440, found 348.1449. HPLC (100% MeOH): t_(R)=4.542 min,97.65%.

Example 20

6-chloro-N⁴-(2-(4-cyclopropylpiperazin-1-yl)-5-nitrophenyl)pyrimidine-4,5-diamine

Following the preparation method in Step 2 of Example 18, morpholine wasreplaced by 1-cyclopropylpiperazine to obtain a yellow solid. Yield58.9%; m.p. 223° C.-225° C.; ¹H NMR (300 MHz, CDCl₃) δ 9.35 (d, J=2.7Hz, 1H), 8.32 (s, 1H), 8.21 (s, 1H), 7.92 (dd, J=8.7, 2.7 Hz, 1H), 3.62(s, 2H), 2.96 (t, J=4.7 Hz, 4H), 2.87 (s, 4H), 1.78-1.71 (m, 1H),0.56-0.51 (m, 2H), 0.49-0.44 (m, 2H). HRMS (ESI): calcd. for m/zC₁₇H₂₁ClN₇O₂, [M+H]⁺ 390.1440, found 390.1446. HPLC (100% MeOH):t_(R)=4.811 min, 99.31%.

Example 21

2-(4-(2-((5-amino-6-chloropyrimidin-4-yl)amino)-4-nitrophenyl)piperazin-1-yl)ethan-1-ol

Following the preparation method in Step 2 of Example 18, morpholine wasreplaced by 2-(piperazin-1-yl)ethan-1-ol to obtain a yellow solid. Yield76.4%; m.p. 202° C.-204° C.; ¹H NMR (300 MHz, CDCl₃) δ 9.33 (s, 1H),8.32 (s, 1H), 8.15 (s, 1H), 7.94 (d, J=8.7 Hz, 1H), 7.27 (s, 1H), 3.70(s, 2H), 3.64-2.46 (m, 2H), 3.02 (s, 4H), 2.78 (s, 4H), 2.68 (s, 2H),2.57 (s, 1H). HRMS (ESI): calcd. for m/z C₁₆H₂₀ClN₇O₃, [M+H]⁺ 394.1389,found 394.1387. HPLC (100% MeOH): t_(R)=7.347 min, 96.12%.

Example 22

6-chloro-N⁴-(5-nitro-2-(4-phenylpiperazin-1-yl)phenyl)pyrimidine-4,5-diamine

Following the preparation method in Step 2 of Example 18, morpholine wasreplaced by 1-phenylpiperazine to obtain a yellow solid. Yield 76.4%;m.p.>250° C.; ¹H NMR (300 MHz, CDCl₃) δ 9.39 (d, J=2.7 Hz, 1H), 8.35 (s,1H), 8.25 (s, 1H), 7.97 (dd, J=8.9, 2.6 Hz, 1H), 7.39-7.28 (m, 3H),7.06-6.90 (m, 3H), 3.54 (s, 2H), 3.44 (t, J=4.9 Hz, 4H), 3.16 (t, J=4.9Hz, 4H). HRMS (ESI): calcd. for m/z C₂₀H₂₀ClN₇O₂, [M+H]⁺ 426.1440, found426.1449. HPLC (100% MeOH): t_(R)=4.905 min, 95.52%.

Example 23

(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(morpholino)methanoneStep 1: Preparation of methyl4′-fluoro-3′-nitro-[1,1′-biphenylyl]-4-carboxylate

4-bromo-1-fluoro-2-nitrobenzene (5.0 g, 22.8 mmol) was dissolved in1,4-dioxane (100 mL), and the solution was added to(4-(methoxylcarbonyl)phenyl)boric acid (27.3 mmol). Cesium carbonate(14.8 g, 45.6 mmol) and a catalytic amount of Pd(PPh₃)₂Cl₂ were added.The mixture was refluxed for 20 hrs under the protection of N₂. Thereaction mixture was filtered to remove the catalyst and cesiumcarbonate, and spin-dried to obtain a crude product, which was purifiedby column chromatography (dichloromethane:methanol=50:1) to obtain awhite solid. Yield 69.9%; m.p. 145° C.-147° C. ¹H NMR (300 MHz, CDCl₃) δ8.31-8.13 (m, 3H), 7.88-7.59 (m, 1H), 8.28 (m, 3H), 7.42-7.36 (m, 1H),3.94 (s, 3H). m/z (EI-MS): 276.2 [M]⁺.

Step 2: Preparation of methyl4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 1 of Example 1,2-F-5-nitrophenylamine was replaced by methyl4′-fluoro-3′-nitro-[1,1′-biphenylyl]-4-carboxylate to obtain a yellowsolid. Yield 93.8%; m.p. 130° C.-132° C.; ¹H NMR (300 MHz, CDCl₃) δ 8.11(dd, J=6.8, 1.9 Hz, 2H), 8.05 (d, J=2.3 Hz, 1H), 7.44 (dd, J=8.6, 2.3Hz, 1H), 7.68-7.61 (m, 2H), 7.21 (d, J=8.6 Hz, 1H), 3.95 (s, 3H), 3.16(t, J=4.7 Hz, 4H), 2.61 (t, J=4.7 Hz, 4H), 2.38 (s, 3H). m/z (EI-MS):356.2 [M]⁺.

Step 3: Preparation of methyl3′-amino-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate

Methyl4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenyl]-4-carboxylate (12.9mmol) was dissolved in methanol, and a catalytic amount of Pd/C wasadded. The mixture was stirred under the protection of H₂ at roomtemperature for 2 hrs. The reaction mixture was filtered and spin-driedto obtain a gray white solid. Yield 90.4%; m.p. 229° C.-231° C.; ¹H NMR(300 MHz, CDCl₃) δ 8.07 (d, J=8.5 Hz, 2H), 7.61 ((dd, J=6.7, 1.8 Hz,2H), 7.09 (d, J=8.5 Hz, 1H), 7.03 (m, 2H), 4.06 (s, 2H), 3.93 (s, 3H),3.01 (t, J=4.4 Hz, 4H), 2.61 (s, 4H), 2.38 (s, 3H). m/z (EI-MS): 326.2[M]⁺.

Step 4: Preparation of methyl3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate

4,6-dichloro-5-nitropyrimidine (7.40 g, 38.11 mmol) was dissolved intetrahydrofuran (50 mL), and then triethyl amine (3.17 mL, 22.86 mmol)was added. A solution of methyl3′-amino-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate intetrahydrofuran was added dropwise at 0° C., heated to room temperatureand stirred for 6 hrs. The reaction mixture was spin-dried to obtain acrude product, which was separated by silica gel column chromatography(dichloromethane:methanol=100:1) to obtain a yellow solid. The yellowsolid was dissolved in ethyl acetate, and then SnCl₂ (3× equiv.) wasadded. and. The mixture was refluxed for 4 hrs, adjusted with asaturated sodium bicarbonate solution until no bubbles were generated,and filtered under suction. The filter cake was washed with ethylacetate (4×20 mL), and the filtrate was extracted with ethyl acetate(4×50 mL). The organic layers were combined, dried, and spin-dried toobtain a gray white solid. Yield 45.7%; m.p. 209° C.-210° C.; ¹H NMR(300 MHz, DMSO-d₆) δ 8.53-8.42 (m, 1H), 8.23 (s, 1H), 7.96-7.90 (m, 3H),7.70 (d, J=8.4 Hz, 2H), 7.46-7.42 (m, 1H), 7.26 (d, J=8.3 Hz, 1H), 5.36(s, 2H), 3.93 (s, 3H), 3.57 (t, J=4.6 Hz, 4H), 2.87 (t, J=4.7 Hz, 4H),2.24 (s, 3H). m/z (EI-MS): 453.2 [M]⁺.

Step 5: Preparation of 3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylic Acid

Methyl 3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate wasdissolved in methanol (50 mL), and then lithium hydroxide (1 M, 10 mL)was added. The mixture was stirred at room temperature for 2 hrs,adjusted with 1M HCl until a large amount of solid precipitated,filtered under suction, and dried, to obtain a white solid. Yield 80.0%;m p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.33-8.28 (m, 2H), 8.10 (s,1H), 7.86-7.84 (m, 2H), 7.80 (s, 1H), 7.13-7.11 (m, 2H), 6.88 (d, J=2.0Hz, 1H), 5.42 (s, 2H), 3.54 (t, J=4.6 Hz, 4H), 2.35 (t, J=4.7 Hz, 4H),2.24 (s, 3H). m z (EI-MS): 439.3 [M]⁺.

Step 6: Preparation of a Target Product:(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(morpholino)methanone

Methyl3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate(0.1 g, 0.26 mmol) was dissolved in DMF (10 mL), and then thencondensing agent BOP (0.22 g, 0.49 mmol) and triethyl amine (0.12 mL,0.88 mmol) were added. The mixture was stirred at room temperature for30 min, and then, morpholine (0.39 mmol) was added. The mixture wasstirred at room temperature for 12 hrs. Then, the reaction mixture waspoured into water (50 mL), and extracted with ethyl acetate (3×50 mL).The organic layer was dried over anhydrous sodium sulfate, filtered,spin-dried, and separated and purified by column chromatography(dichloromethane:methanol=50:1) to obtain a white solid. Yield 67.8%;m.p. 225° C.-227° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.45 (d, J=13.6 Hz,2H), 7.90 (s, 1H), 7.69 (d, J=7.9 Hz, 2H), 7.49 (d, J=7.7 Hz, 2H), 7.42(d, J=8.4 Hz, 1H), 7.27 (d, J=8.2 Hz, 1H), 5.39 (s, 2H), 3.61 (s, 4H),3.35 (s, 4H), 2.98-2.90 (s, 4H), 2.64 (s, 4H), 2.34 (s, 3H). HRMS (ESI):calcd. for m/z C₂₆H₃₀ClN₇O₂, [M+H]⁺ 508.2222, found 508.2223. HPLC (80%methanol in water): t_(R)=3.507 min, 98.81%.

Example 24

3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-N-(2-morpholinoethyl)-[1,1′-biphenyl]-zole-4-carboxamide

Following the preparation method in Step 6 of Example 23, morpholine wasreplaced by 2-morpholinoethyl-1-amine to obtain a white solid. Yield55.8%; m.p. 230° C.-232° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.45 (s, 1H),8.27-8.22 (m, 2H), 7.92-7.90 (m, 3H), 7.71 (d, J=8.0 Hz, 2H), 7.45 (d,J=8.2 Hz, 1H), 7.26 (d, J=8.4 Hz, 1H), 5.39 (s, 2H), 3.58 (s, 4H),3.40-3.34 (m, 4H), 2.90 (s, 4H), 2.61 (s, 4H), 2.44 (s, 4H), 2.31 (s,3H). HRMS (ESI): calcd. for m/z C₂₈H₃₅ClN₈O₂, [M+H]⁺ 551.2644, found551.2639. HPLC (80% methanol in water): t_(R)=3.196 min, 98.91%.

Example 25

3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-N-(3-morpholinopropyl)-[1,1′-biphenyl]-zole-4-carboxamide

Following the preparation method in Step 6 of Example 23, morpholine wasreplaced by 3-morpholinopropyl-1-amine to obtain a white solid. Yield48.9%; m.p. 210° C.-212° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.53-8.42 (m,1H), 8.28 (d, J=2.8 Hz, 1H), 8.23 (s, 1H), 7.96-7.90 (m, 3H), 7.70 (d,J=8.4 Hz, 2H), 7.46-7.42 (m, 1H), 7.26 (d, J=8.3 Hz, 1H), 5.36 (s, 2H),3.57 (t, J=4.6 Hz, 4H), 3.32-3.30 (m, 2H), 2.87 (t, J=4.7 Hz, 4H),2.51-2.49 (m, 4H), 2.37-2.32 (m, 6H), 2.24 (s, 3H). HRMS (ESI): calcd.for m/z C₂₉H₃₁ClN₈O₂, [M+H]⁺ 565.2801, found 565.2801. HPLC (80%methanol in water): t_(R)=3.974 min, 96.00%.

Example 26

3″-((5-amino-6-chloropyrimidin-4-yl) amino)-N,N-dimethyl-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxamide

Following the preparation method in Step 6 of Example 23, morpholine wasreplaced by dimethyl amine to obtain a white solid. Yield 78.9%; m.p.142° C.-145° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.24 (s, 1H), 7.70 (s, 2H),7.56-7.41 (m, 5H), 7.02 (s, 1H), 5.57 (s, 2H), 3.19-3.97 (m, 10H), 2.75(s, 4H), 2.43 (s, 3H). HRMS (ESI): calcd. for m/z C₂₄H₂₈ClN₇O, [M+H]⁺466.2117, found 466.2121. HPLC (80% methanol in water): t_(R)=2.489 min,95.56%.

Example 27

(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(4-methylpiperazin-1-yl)methanone

Following the preparation method in Step 6 of Example 23, morpholine wasreplaced by 1-methylpiperazine to obtain a white solid. Yield 56.9%;m.p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.54 (d, J=13.6 Hz, 2H), 7.90(s, 1H), 7.68 (d, J=7.9 Hz, 2H), 7.45 (d, J=7.7 Hz, 2H), 7.39 (d, J=8.4Hz, 1H), 7.27 (d, J=8.1 Hz, 1H), 5.35 (s, 2H), 3.61 (s, 4H), 3.35 (s,4H), 2.98-2.90 (s, 4H), 2.64 (s, 4H), 2.34 (s, 3H), 2.30 (s, 3H). HRMS(ESI): calcd. for m/z C₂₇H₃₃ClN₈O, [M+H]⁺ 521.2937, found 521.2937. HPLC(80% methanol in water): t_(R)=4.094 min, 98.10%.

Example 28

3″-((5-amino-6-chloropyrimidin-4-yl)amino)-N-(3-(dimethylamino)propyl)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxamide

Following the preparation method in Step 6 of Example 23, morpholine wasreplaced by N¹, N¹-dimethyl-1,3-diamine to obtain a white solid. Yield38.9%; m.p. 232° C.-234° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.66 (s, 1H),8.26-8.20 (m, 2H), 7.93-7.88 (m, 3H), 7.73 (d, J=9.8 Hz, 2H), 7.47 (s,1H), 7.29 (s, 1H), 5.44 (s, 2H), 3.08-2.90 (m, 8H), 2.77 (s, 6H), 2.50(s, 4H), 1.90 (s, 4H), 1.28-1.23 (m, 2H). HRMS (ESI): calcd. for m/zC₂₇H₃₆ClN₈O, [M+H]⁺ 523.2695, found 523.2695. HPLC (90% methanol inwater): t_(R)=3.409 min, 98.88%.

Example 29

3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-N-(2-(pyrrolidin-1-yl)ethyl)-[11,1′-biphenylyl]-4-carboxamide

Following the preparation method in Step 6 of Example 23, morpholine wasreplaced by 2-(pyrimidin-1-yl)ethan-1-amine to obtain a white solid.Yield 37.8%; m.p. 241° C.-243° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.67 (s,1H), 8.25-8.22 (m, 2H), 7.93-7.88 (m, 3H), 7.73 (d, J=9.8 Hz, 2H), 7.47(s, 1H), 7.29 (s, 1H), 5.64 (s, 2H), 3.64-3.62 (m, 2H), 3.38-3.34 (m,6H), 3.12 (s, 4H), 2.54 (s, 2H), 2.48 (s, 2H), 2.36 (s, 3H), 1.93 (s,4H). HRMS (ESI): calcd. for m/z C₂₈H₃₅ClN₈O, [M+H]⁺ 535.2695, found535.2690. HPLC (100% methanol): t_(R)=7.516 min, 98.64%.

Example 30

3″-((5-amino-6-chloropyrimidin-4-yl)amino)-N-(3-hydroxylpropyl)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-zole-4-carboxamide

Following the preparation method in Step 6 of Example 23, morpholine wasreplaced by 3-aminopropan-1-ol to obtain a white solid. Yield 50.7%;m.p. 189° C.-192° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.45 (d, J=7.2 Hz,1H), 8.30-8.19 (m, 2H), 7.93-7.90 (m, 3H), 7.72-7.69 (m, 2H), 7.43 (d,J=8.4 Hz, 1H), 7.26 (d, J=8.4 Hz, 1H), 5.37 (s, 2H), 4.49-4.47 (m, 1H),3.51-3.45 (m, 4H), 2.88 (s, 4H), 2.52-2.50 (m, 4H), 2.27 (s, 3H),1.74-1.64 (m, 2H). HRMS (ESI): calcd. for m/z C₂₅H₃₁ClN₇O₂, [M+H]⁺496.2222, found 496.2217. HPLC (80% methanol in water): t_(R)=2.384 min,96.63%

Example 31

3″-((5-amino-6-chloropyrimidin-4-yl)amino)-N-isopropyl-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxamide

Following the preparation method in Step 6 of Example 23, morpholine wasreplaced by propan-2-amine to obtain a white solid. Yield 63.8%; m.p.184° C.-186° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.27-8.22 (m, 2H), 7.91 (d,J=8.1 Hz, 2H), 7.71 (s, 1H), 7.59 (d, J=8.0 Hz, 2H), 7.43-7.30 (m, 2H),7.04 (d, J=8.2 Hz, 1H), 5.54 (s, 2H), 4.12 (s, 1H), 3.33 (s, 4H), 3.07(s, 4H), 2.82 (s, 3H), 1.18 (s, 6H). HRMS (ESI): calcd. for m/zC₂₅H₃₀ClN₇O, [M+H]⁺ 480.2279, found 480.2276. HPLC (80% methanol inwater): t_(R)=3.601 min, 96.77%.

Example 32

Step 1: Preparation of 1-(4-bromo-2-nitrophenyl)-4-methylpiperazine

2-fluoro-5-bromonitrobenzene (10.0 g, 45.4 mmol) was dissolved in DMF(20 mL), and DIPEA (9.5 mL, 54.6 mmol) and 1-methylpiperazine (6.6 mL,54.6 mmol) were added. The mixture was stirred at 80° C. for 8 hrs. Thereaction mixture was poured into 100 mL of water, and extracted withethyl acetate (3×100 mL). The organic layer was dried over anhydroussodium sulfate, filtered, and spin-dried to obtain a yellow solid. Yield95.0%; ¹H NMR (300 MHz, CDCl₃) δ 7.91 (d, J=2.4 Hz, 1H), 7.57 (dd,J=8.8, 2.4 Hz, 1H), 7.04 (d, J=8.8 Hz, 1H), 3.08 (t, J=4.9 Hz, 4H), 2.58(t, J=4.9 Hz, 4H), 2.37 (s, 3H). m/z (EI-MS): 300.0 [M]⁺.

Step 2: Preparation of 5-bromo-2-(4-methylpiperazin-1-yl)phenylamine

1-(4-bromo-2-nitrophenyl)-4-methylpiperazine (12.9 g, 43.14 mmol) wasdissolved in methanol (100 mL), and a catalytic amount of Pd/C wasadded. The mixture was kept under the protection of hydrogen at roomtemperature for 2 hrs, filtered, and spin-dried to obtain a yellowsolid. Yield 89%; m.p. 165-167° C.; ¹H NMR (300 MHz, CDCl₃) δ6.84-6.79(m, 3H), 4.01 (s, 2H), 2.89 (s, 4H), 2.45 (s, 4H), 2.35 (s, 3H). m/z(EI-MS): 270.0 [M]⁺.

Step 3: Preparation of N⁴-(5-bromo-2-(4-methylpiperazin-1-yl)phenyl)-6-chloropyrimidine-4,5-diamine

Following the preparation method in Step 4 of Example 23, methyl3′-amino-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate wasreplaced by 5-bromo-2-(4-methylpiperazin-1-yl) phenylamine, to obtain awhite solid. Yield 72.5%; m.p. 196-198° C.; ¹H NMR (300 MHz, CDCl₃) δ8.26 (s, 1H), 6.84-6.79 (m, 3H), 8.10 (s, 1H), 5.34 (s, 2H), 2.89 (s,4H), 2.45 (s, 4H), 2.34 (s, 3H). m/z (EI-MS): 397.1 [M]⁺.

Step 4: Preparation of a Target ProductN⁴-(4′-amino-4-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-3-yl)-6-chloropyrimidine-e-4,5-diamine

N⁴-(5-bromo-2-(4-methylpiperazin-1-yl)phenyl)-6-chloropyrimidine-4,5-diamine(1.0 g, 2.5 mmol) was dissolved in 1,4-dioxane (30 mL), and then4-aminophenylboric acid (2.9 mmol), cesium carbonate (1.6 g, 5 mmol),and a catalytic amount of Pd(PPh₃)₂Cl₂ were added. The mixture wasrefluxed under the protection of nitrogen for 20 hrs, and filtered toremove the catalyst and cesium carbonate. The filtrate was spin-dried,and separated and purified by column chromatography(dichloromethane:methanol=50:1) to obtain a light yellow solid. Yield67.3%; m.p. 212° C.-214° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.16 (d, J=8.1Hz, 2H), 7.90 (s, 1H), 7.65 (d, J=8.2 Hz, 2H), 7.55 (d, J=8.4 Hz, 2H),7.37 (d, J=8.4 Hz, 1H), 7.26 (d, J=8.2 Hz, 1H), 5.43 (s, 2H), 5.15 (s,2H), 3.17 (s, 4H), 3.06 (s, 4H), 2.75 (s, 3H). m/z (EI-MS): 410.2 [M]⁺.HPLC (100% methanol): t_(R)=7.489 min, 95.67%.

Example 33

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)tetrahydro-2H-pyran-4-carboxamide

Tetrahydro-2H-pyran-4-carboxylic acid (0.35 mmol) was dissolved in DMF(8 mL), and then BOP (0.26 g, 0.59 mmol) and triethyl amine (0.1 mL,0.88 mmol) were added. The mixture was stirred at room temperature for30 min, and then the title compound (0.12 g, 0.29 mmol) of Example 32was added. The mixture was stirred at room temperature for 12 h. Thereaction mixture was poured into 50 mL of water, extracted with ethylacetate (3×50 mL), dried over anhydrous sodium sulfate, filtered,spin-dried, and separated and purified by silica gel columnchromatography (dichloromethane:methanol=50:1) to obtain a white solid.Yield 78.5%; m.p. 207° C.-209° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.99 (s,1H), 8.21 (s, 1H), 7.90 (s, 1H), 7.68 (dd, J=8.4, 5.9 Hz, 3H), 7.57-7.54(m, 2H), 7.38-7.35 (m, 1H), 5.38 (s, 2H), 3.94-3.90 (m, 3H), 3.37-3.35(m, 2H), 2.92 (s, 4H), 2.76 (s, 4H), 2.43 (s, 3H), 1.72-1.69 (m, 4H).HRMS (ESI): calcd. for m/z C₂₇H₃₂ClN₇O₂, [M+H]⁺ 522.2379, found522.2377. HPLC (80% methanol in water): t_(R)=4.280 min, 95.50%.

Example 34

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)-1-methylpiperidin-4-carboxamide

Following Example 33, tetrahydro-2H-pyran-4-carboxylic acid was replacedby 1-methylpiperidin-4-carboxylic acid, to obtain a white solid. Yield63.6%; m.p. 157° C.-160° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 10.19 (s, 1H),8.22-8.19 (m, 2H), 7.95 (s, 1H), 7.69 (d, J=8.4 Hz, 2H), 7.57 (d, J=8.7Hz, 2H), 7.36 (d, J=8.4 Hz, 1H), 7.24 (d, J=8.1 Hz, 1H), 5.42 (s, 2H),2.99-2.90 (m, 5H), 2.85 (s, 4H), 2.73 (s, 3H), 2.53 (s, 4H), 2.49 (s,3H), 2.03-1.99 (m, 4H). HRMS (ESI): calcd. for m/z C₂₈H₃₆ClN₈O, [M+H]⁺535.2695, found 535.2710. HPLC (80% methanol in water): t_(R)=4.518 min,98.25%.

Example 35

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)cyclohexanecarboxamide

Following Example 33, tetrahydro-2H-pyran-4-carboxylic acid was replacedby cyclohexane-carboxylic acid, to obtain a white solid. Yield 80.1%;m.p. 256° C.-258° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.91 (s, 1H), 8.18 (d,J=8.2 Hz, 2H), 7.90 (s, 1H), 7.69 (d, J=8.3 Hz, 2H), 7.55 (d, J=8.4 Hz,2H), 7.37 (d, J=8.4 Hz, 1H), 7.26 (d, J=8.2 Hz, 1H), 5.43 (s, 2H), 3.17(s, 4H), 3.06 (s, 4H), 2.75 (s, 3H), 2.34-2.30 (m, 1H), 1.76 (t, J=12.5Hz, 4H), 1.42 (t, J=11.6 Hz, 2H), 1.33-1.09 (m, 4H). HRMS (ESI): calcd.for m/z C₂₈H₃₄ClN₇O, [M+H]⁺ 520.2586, found 520.2584. HPLC (90% methanolin water): t_(R)=4.003 min, 97.87%.

Example 36

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)butyramide

Following Example 33, tetrahydro-2H-pyran-4-carboxylic acid was replacedby n-butanoic acid, to obtain a white solid. Yield 79.7%; m.p. 196°C.-198° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 10.00 (s, 1H), 8.21-8.16 (m,2H), 7.90 (d, J=6.0 Hz, 1H), 7.71-7.69 (m, 2H), 7.59-7.54 (m, 2H), 7.38(d, J=7.8 Hz, 1H), 7.27 (d, J=8.2 Hz, 1H), 5.44 (s, 2H), 3.51-3.46 (m,4H), 3.27-3.16 (m, 4H), 2.88 (s, 3H), 2.32-2.27 (m, 2H), 1.68-1.58 (m,2H), 0.96-0.89 (m, 3H). HRMS (ESI): calcd. for m/z C₂₅H₃₁ClN₇O, [M+H]⁺480.2273, found 480.2274. HPLC (80% methanol in water): t_(R)=4.696 min,96.97%.

Example 37

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)benzenecarboxamide

Following Example 33, tetrahydro-2H-pyran-4-carboxylic acid was replacedby benzoic acid, to obtain a white solid. Yield 77.9%; m.p.>250° C.; ¹HNMR (300 MHz, DMSO-d₆) δ 10.00 (s, 1H), 8.21-8.16 (m, 2H), 7.90 (d,J=6.0 Hz, 1H), 7.71-7.69 (m, 2H), 7.59-7.54 (m, 2H), 7.38 (d, J=7.8 Hz,1H), 7.27 (d, J=8.2 Hz, 1H), 5.44 (s, 2H), 3.51-3.46 (m, 4H), 3.27-3.16(m, 4H), 2.88 (s, 3H), 2.32-2.27 (m, 2H), 1.68-1.58 (m, 2H), 0.96-0.89(m, 3H). HRMS (ESI): calcd. for m/z C₂₈H₂₈ClN₇O, [M+H]⁺ 514.2117, found514.2115. HPLC (90% methanol in water): t_(R)=3.889 min, 95.09%.

Example 38

(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(4-methylpiperazin-1-yl)methanone

Following the preparation method in Step 4 of Example 32,4-aminophenylboric acid was replaced by 2-F-4-carboxylphenylboric acid,to obtain a white intermediate. Following the preparation method in Step6 of Example 23, morpholine was replaced by 1-methylpiperazine to obtaina white solid. Yield 43.1%; m.p. 182° C.-184° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 8.12 (s, 1H), 7.80 (s, 1H), 7.68-7.65 (m, 2H), 7.60 (dd,J=7.5, 2.0 Hz, 1H), 7.10 (dd, J=7.5, 2.0 Hz, 1H), 6.93 (d, J=7.3 Hz,1H), 6.82 (d, J=2.0 Hz, 1H), 5.36 (s, 2H), 3.55 (t, J=5.1 Hz, 4H), 3.30(t, J=5.1 Hz, 4H), 2.85 (t, J=4.7 Hz, 4H), 2.53 (s, 4H), 2.34 (s, 3H),2.32 (s, 3H). HRMS (ESI): calcd. for m/z C₂₇H₃₂ClFN₈O, [M+H]⁺ 539.2444,found 539.2443. HPLC (80% methanol in water): t_(R)=4.156 min, 95.21%.

Example 39

N-(3′-amino-4-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-3-yl)-6-chloropyrimidine-4,5-diamine

Following the preparation method in Step 4 of Example 32,4-aminophenylboric acid was replaced by 3-aminophenylboric acid, toobtain a white solid. Yield 69.4%; m.p. 215° C.-217° C.; ¹H NMR (300MHz, DMSO-d₆) δ 8.23 (s, 1H), 8.17 (s, 1H), 7.90 (s, 1H), 7.23 (d, J=6.4Hz, 2H), 7.07 (d, J=7.7 Hz, 1H), 6.80 (s, 1H), 6.73 (d, J=7.6 Hz, 1H),6.53 (d, J=8.0 Hz, 1H), 5.36 (s, 2H), 5.17 (s, 2H), 2.85 (s, 4H), 2.61(s, 4H), 2.25 (s, 3H). HRMS (ESI): calcd. for m/z C₂₁H₂₄ClN₇, [M+H]⁺410.1854, found 410.1852. HPLC (100% methanol): t_(R)=7.491 min, 95.66%.

Example 40

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-3-yl)tetrahydro-2H-pyran-4-carboxamide

Following the preparation method in Example 33, the title compound ofExample 32 was replaced by the title compound of Example 39, to obtain awhite solid. Yield 65.0%; m.p. 212° C.-214° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 9.99 (s, 1H), 8.24 (s, 1H), 8.19 (s, 1H), 7.89 (s, 2H), 7.59(d, J=8.0 Hz, 1H), 7.44-7.19 (m, 4H), 5.38 (s, 2H), 3.91 (d, J=10.9 Hz,2H), 3.47-3.35 (m, 3H), 2.86 (s, 4H), 2.54 (m, 4H), 2.24 (s, 3H), 1.70(s, 4H). HRMS (ESI): calcd. for m/z C₂₇H₃₂ClN₇O₂, [M+H]⁺ 522.2379, found522.2368. HPLC (100% methanol): t_(R)=7.105 min, 98.72%.

Example 41

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-3-yl)-1-methylpiperidin-4-carboxamide

Following the preparation method in Example 34, the title compound ofExample 32 was replaced by the title compound of Example 39, to obtain awhite solid. Yield 56.9%; m.p. 158° C.-160° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 9.95 (s, 1H), 8.24 (s, 1H), 8.18 (s, 1H), 7.89 (s, 2H), 7.59(d, J=8.3 Hz, 1H), 7.39-7.19 (m, 4H), 5.38 (s, 2H), 2.86-2.83 (m, 4H),2.80 (s, 1H), 2.57-2.51 (m, 4H), 2.22 (s, 3H), 2.15 (s, 3H), 1.89-1.82(m, 2H), 1.79-1.58 (m, 4H), 1.22 (s, 2H). HRMS (ESI): calcd. for m/zC₂₈H₃₆ClN₈O, [M+H]⁺ 535.2695, found 535.2702. HPLC (100% methanol):t_(R)=7.252 min, 95.45%.

Example 42

6-chloro-N-(4′-methoxyl-4-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-3-yl)pyrimidine-4,5-diamine

Following the preparation method in Step 4 of Example 32,4-aminophenylboric acid was replaced by 4-methoxylphenylboric acid, toobtain a white solid. Yield 59.9%; m.p. 211° C.-213° C.; ¹H NMR (300MHz, DMSO-d₆) δ 8.24 (s, 2H), 7.90 (s, 1H), 7.57 (d, J=7.9 Hz, 2H),7.40-7.35 (m, 3H), 7.24 (d, J=8.3 Hz, 1H), 5.37 (s, 2H), 5.22 (s, 1H),4.52 (s, 2H), 2.85 (t, J=4.5 Hz, 4H), 2.53 (s, 4H), 2.24 (s, 3H). m z(EI-MS): 425.2 [M]⁺. HPLC (80% methanol in water): t_(R)=4.590 min,99.44%.

Example 43

6-chloro-N⁴-(5-(furan-3-yl)-2-(4-methylpiperazin-1-yl)phenyl)pyrimidine-4,5-diamine

Following the preparation method in Step 4 of Example 32,4-aminophenylboric acid was replaced by 3-furylphenylboric acid, toobtain a white solid. Yield 70.1%; m.p. 164° C.-166° C.; ¹H NMR (300MHz, DMSO-d₆) δ 8.23 (s, 1H), 7.90 (s, 1H), 7.54 (d, J=7.5 Hz, 1H), 7.43(s, 1H), 7.07 (d, J=7.7 Hz, 1H), 6.91 (d, J=7.4 Hz, 1H), 6.82 (s, 1H),6.53 (d, J=8.0 Hz, 1H), 5.36 (s, 2H), 2.85 (s, 4H), 2.61 (s, 4H), 2.25(s, 3H). HRMS (ESI): calcd. for m/z C₁₉H₂₂ClN₆O, [M+H]⁺ 385.1538, found385.1538. HPLC (90% methanol in water): t_(R)=6.859 min, 96.26%.

Example 44

6-chloro-N⁴-(2-(4-methylpiperazin-1-yl)-5-(pyrimidine-5-yl)phenyl)pyrimidine-4,5-diamine

Following the preparation method in Step 4 of Example 32,4-aminophenylboric acid was replaced by 4-pyrimidinylphenylboric acid,to obtain a white solid. Yield 45.8%; m.p. 189° C.-191° C.; ¹H NMR (300MHz, DMSO-d₆) δ 9.17 (s, 1H), 9.10 (s, 2H), 8.57 (s, 1H), 7.80 (s, 1H),7.10 (dd, J=7.5, 2.0 Hz, 1H), 6.99 (d, J=7.4 Hz, 1H), 6.82 (d, J=2.0 Hz,1H), 5.32 (s, 2H), 2.85 (s, 4H), 2.54 (s, 4H), 2.34 (s, 3H). HRMS (ESI):calcd. for m/z C₁₉H₂₂ClN₈, [M+H]⁺ 397.1651, found 397.1653. HPLC (80%methanol in water): t_(R)=3.973 min, 97.50%.

Example 45

6-chloro-N⁴-(2-(4-methylpiperazin-1-yl)-5-(pyridin-4-yl)phenyl)pyrimidine-4,5-diamine

Following the preparation method in Step 4 of Example 32,4-aminophenylboric acid was replaced by 4-pyridinylphenylboric acid, toobtain a white solid. Yield 44.2%; m.p. 202° C.-204° C.; ¹H NMR (300MHz, DMSO-d₆) δ 8.83 (s, 1H), 8.69 (d, J=5.1 Hz, 2H), 7.80 (s, 1H), 7.75(s, 2H), 7.10 (dd, J=7.5, 2.0 Hz, 1H), 6.94 (d, J=7.5 Hz, 1H), 6.82 (s,1H), 5.54 (s, 2H), 3.30 (t, J=5.1 Hz, 4H), 2.85 (t, J=4.7 Hz, 4H), 2.54(s, 4H), 2.34 (s, 3H), 2.32 (s, 3H). m/z (EI-MS): 411.1 [M]⁺. HPLC (100%methanol): t_(R)=7.074 min, 96.00%.

Example 46

Methyl 1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylateStep 1: Preparation of 4-(4-methylpiperazin-1-yl)-3-nitrophenylamine

4-F-3-nitrophenylamine (5 g, 32 mmol) was dissolved in acetonitrile (50mL), and then DIPEA (7.9 mL, 47.8 mmol) and 1-methylpiperazine (5.3 mL,67.8 mmol) were added. The mixture was refluxed for 12 hrs. The reactionmixture was spin-dried to obtain a crude red solid, which was washedwith acetonitrile, filtered under suction, and dried. Yield 95.5%;m.p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 7.06 (d, J=8.6 Hz, 1H), 6.76(s, 1H), 6.69 (d, J=8.5 Hz, 1H), 5.34 (s, 2H), 2.70 (t, J=4.4 Hz, 4H),2.27 (s, 4H), 2.09 (s, 3H). m/z (EI-MS): 237.1 [M]⁺.

Step 2: Preparation of 1-(4-azido-2-nitrophenyl)-4-methylpiperazine

4-(4-methylpiperazin-1-yl)-3-nitrophenylamine (5.0 g, 21.25 mmol) wasdissolved in 2M HCl. An aqueous solution (12 mL) of NaNO₂ (2.2 g, 31.8mmol) was added dropwise in an ice bath. The mixture was stirred underan ice bath condition for 30 min. Then, sodium azide (2.8 g, 42.5 mmol)was added. The mixture was heated to room temperature, and stirred for 2hrs. The reaction mixture was adjusted to pH=9 to 10 with 2 M NaOH untila reddish brown solid precipitated, which was filtered and dried. Yield87.7%; m.p. 88-91° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 7.48 (d, J=2.2 Hz,1H), 7.34-7.20 (m, 2H), 2.85 (t, J=4.7 Hz, 4H), 2.31 (t, J=4.8 Hz, 4H),2.11 (s, 3H). m/z (EI-MS): 262.1 [M]⁺.

Step 3: Preparation of methyl1-(4-(4-methylpiperazin-1-yl)-3-nitrophenyl)-1H-1,2,3-triazole-4-carboxylate

1-(4-azido-2-nitrophenyl)-4-methylpiperazine (2.0 g, 7.6 mmol) wasdissolved in methanol (50 mL), and methyl propiolate (1.8 g, 22.8 mmol)was added. Then, CuI (0.14 g, 0.76 mmol) and DIPEA (1.2 mL, 7.6 mmol)were added. The mixture was refluxed for 48 hrs, and filtered to removeCuI. The filtrate was spin-dried to obtain a crude product, which waswashed with ethyl acetate to obtain a reddish brown solid. Yield 61.7%;m.p. 159-161° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.45 (s, 1H), 8.36 (d,J=2.7 Hz, 1H), 8.11-8.01 (m, 1H), 7.42 (d, J=9.1 Hz, 1H), 3.80 (s, 3H),2.99 (t, J=5.4 Hz, 4H), 2.35 (t, J=5.2 Hz, 4H), 2.13 (s, 3H). m/z(EI-MS): 369.2 [M+Na]⁺.

Step 4: Preparation of methyl1-(3-amino-4-(4-methylpiperazin-1-yl)phenyl)-1H-1, 2,3-triazole-4-carboxylate

Methyl1-(4-(4-methylpiperazin-1-yl)-3-nitrophenyl)-1H-1,2,3-triazole-4-carboxylate(1.7 g, 4.9 mmol) was dissolved in methanol (50 mL), and a catalyticamount of Pd/C was added. The mixture was stirred under the protectionof hydrogen at room temperature for 6 hrs, and filtered to remove Pd/C.The filtrate was spin-dried to obtain a pink solid. Yield 85.3%; m.p.194-197° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.28 (s, 1H), 7.28 (d, J=1.9Hz, 1H), 7.07 (d, J=1.9 Hz, 2H), 5.15 (s, 2H), 3.90 (s, 3H), 2.87 (t,J=4.5 Hz, 4H), 2.53 (br s, 4H), 2.26 (s, 3H). m/z (EI-MS): 317.2 [M]⁺.

Step 5: Preparation of a Target Product: methyl1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate

Following the preparation method in Step 4 of Example 23, methyl3′-amino-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate wasreplaced by methyl1-(3-amino-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate,to obtain a beige solid. Yield 65.9%; m.p. 231° C.-233° C.; ¹H NMR (300MHz, DMSO-d₆) δ 9.46 (s, 1H), 8.62 (s, 1H), 8.34 (s, 1H), 7.95 (s, 1H),7.64 (d, J=8.6 Hz, 1H), 7.37 (d, J=8.7 Hz, 1H), 5.44 (s, 2H), 3.89 (s,3H), 2.88 (s, 4H), 2.51 (s, 4H), 2.23 (s, 3H). HRMS (ESI): calcd. form/z C₁₉H₂₂ClFN₉O₂, [M+H]⁺ 444.1657, found 444.1660. HPLC (90% methanolin water): t_(R)=3.775 min, 97.63%.

Example 47

(1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-yl)(morpholino)methanone Step 1: Methyl1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate

Following the preparation method in Step 5 of Example 23, methyl3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylatewas replaced by methyl1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate,to obtain a beige solid. Yield 87.6%; m.p. 232° C.-234° C.; ¹H NMR (300MHz, DMSO-d₆) δ 12.20 (s, 1H), 9.36 (s, 1H), 8.62 (s, 1H), 8.34 (s, 1H),7.95 (s, 1H), 7.63 (d, J=8.7 Hz, 1H), 7.38 (d, J=8.7 Hz, 1H), 5.47 (s,2H), 2.90 (s, 4H), 2.53 (s, 4H), 2.34 (s, 3H). HRMS (ESI): calcd. form/z C₁₈H₂₀ClFN₉O₂, [M+H]⁺ 430.1488, found 430.1492. HPLC (90% methanolin water): t_(R)=3.548 min, 99.16%.

Step 2: Preparation of a Target Product:(1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-yl)(morpholino)methanone

Following the preparation method in Step 6 of Example 23,3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylicacid was replaced by1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylicacid, to obtain a beige solid. Yield 68.3%; m.p. 200° C.-202° C.; ¹H NMR(300 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.60 (s, 1H), 8.53 (t, J=5.8 Hz, 1H),8.34 (s, 1H), 7.94 (s, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.37 (d, J=8.6 Hz,1H), 5.45 (s, 2H), 3.58 (t, J=4.5 Hz, 4H), 3.45-3.36 (m, 4H), 2.91 (t,J=4.5 Hz, 4H), 2.60 (s, 4H), 2.44 (s, 4H), 2.30 (s, 3H). HRMS (ESI):calcd. for m/z C₂₄H₃₂ClFN₁O₂, [M+H]⁺ 542.2502, found 542.2500. HPLC (80%methanol in water): t_(R)=4.612 min, 99.65%.

Example 48

1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-N-(3-morpholinopropyl)-1H-1,2, 3-triazole-4-carboxamide

Following the preparation method in Example 24,3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylicacid was replaced by1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylicacid, to obtain a beige solid. Yield 65.9%; m.p. 203° C.-205° C.; ¹H NMR(300 MHz, DMSO-d₆) δ 9.19 (s, 1H), 8.82 (t, J=5.6 Hz, 1H), 8.59 (s, 1H),8.34 (s, 1H) 7.94 (s, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.36 (d, J=8.7 Hz,1H), 5.45 (s, 2H), 3.61 (s, 4H), 3.36-3.33 (m, 4H), 2.90 (s, 4H), 2.58(s, 4H), 2.40 (s, 4H), 2.28 (s, 3H), 1.73-1.68 (m, 2H). HRMS (ESI):calcd. for m/z C₂₅H₃₄ClFN₁O₂, [M+H]⁺ 556.2660, found 556.2662. HPLC (80%methanol in water): t_(R)=4.457 min, 98.27%.

Example 49

(1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-yl)(4-methylpiperazin-1-yl)methanone

Following the preparation method in Example 25,3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylic acidwas replaced by1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylicacid, to obtain a beige solid. Yield 46.2%; m.p. 225° C.-228° C.; ¹H NMR(300 MHz, DMSO-d₆) δ9.19 (s, 1H), 8.72 (s, 1H), 8.52 (s, 1H), 8.34 (s,1H) 7.94 (s, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.37 (d, J=8.7 Hz, 1H), 5.45(s, 2H), 3.76 (s, 4H), 3.39 (s, 4H), 2.93 (s, 4H), 2.68 (s, 4H) 2.22 (s,3H), 2.15 (s, 3H). HRMS (ESI): calcd. for m/z C₂₃H₃₁ClFN₁₁O, [M+H]⁺512.2396, found 512.2397. HPLC (90% methanol in water): t_(R)=3.707 min,98.00%.

Example 50

1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-N,N-dimethyl-1H-1,2,3-triazole-4-carboxamide

Following the preparation method in Example 27,3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylicacid was replaced by1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylicacid, to obtain a beige solid. Yield 66.6%; m.p. 215° C.-217° C.; ¹H NMR(300 MHz, DMSO-d₆) δ 9.84 (s, 1H), 9.18 (s, 1H), 8.62 (s, 1H), 8.27 (s,1H) 7.95 (s, 1H), 7.68 (s, 1H), 7.44 (s, 1H), 5.52 (s, 2H), 3.58 (s,4H), 3.30 (s, 6H), 3.04 (s, 4H), 2.88 (s, 3H). HRMS (ESI): calcd. form/z C₂₀H₂₅ClFN₁₀O, [M+H]⁺ 457.1974, found 457.1966. HPLC (80% methanolin water): t_(R)=4.203 min, 98.52%.

Example 51

1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-N, N-dimethyl-1H-1,2,3-triazole-4-carboxamide

Following the preparation method in Example 26,3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylicacid was replaced by1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylicacid, to obtain a beige solid. Yield 66.6%; m.p. 215° C.-217° C.; ¹H NMR(300 MHz, DMSO-d₆) δ 9.84 (s, 1H), 9.18 (s, 1H), 8.62 (s, 1H), 8.27 (s,1H) 7.95 (s, 1H), 7.68 (s, 1H), 7.44 (s, 1H), 5.52 (s, 2H), 3.58 (s,4H), 3.30 (s, 6H), 3.04 (s, 4H), 2.88 (s, 3H). HRMS (ESI): calcd. form/z C₂₀H₂₅ClFN₁₀O, [M+H]⁺ 457.1974, found 457.1966. HPLC (80% methanolin water): t_(R)=4.203 min, 98.52%.

Example 52

1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-N-(2-(dimethylamino)ethyl)-1H-1,2,3-triazole-4-carboxamide

Following the preparation method in Example 28,3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylicacid was replaced by1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylicacid, to obtain a beige solid. Yield 39.9%; m.p. 149° C.-152° C.; ¹H NMR(300 MHz, DMSO-d₆) δ 9.84 (s, 1H), 9.18 (s, 1H), 8.62 (s, 1H), 8.54 (s,1H), 7.95 (s, 1H), 7.68 (d, J=8.7 Hz, 1H), 7.44 (d, J=8.7 Hz, 1H), 5.46(s, 2H), 3.08-2.90 (m, 6H), 2.77 (s, 6H), 2.50 (s, 4H), 1.90 (s, 4H),1.28-1.23 (m, 2H). HRMS (ESI): calcd. for m/z C₂₂H₃₀ClFN₁₁O, [M+H]⁺500.2402, found 500.2404. HPLC (80% methanol in water): t_(R)=4.157 min,98.88%.

Example 53

1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-N-(2-(pyrrolidin-1-yl)ethyl)-1H-1,2, 3-triazole-4-carboxamide

Following the preparation method in Example 29,3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylicacid was replaced by1-(3-((5-amino-6-chloropyrimidin-4-yl)amino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-carboxylicacid, to obtain a beige solid. Yield 40.7%; m.p. 172° C.-175° C.; ¹H NMR(300 MHz, DMSO-d₆) δ 9.19 (s, 1H), 8.72 (s, 1H), 8.52 (s, 1H), 8.34 (s,1H) 7.94 (s, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.37 (d, J=8.7 Hz, 1H), 5.45(s, 2H), 3.64-3.62 (m, 2H), 3.38-3.34 (m, 6H), 3.12 (s, 4H), 2.54 (s,2H), 2.48 (s, 2H), 2.36 (s, 3H), 1.93 (s, 4H). HRMS (ESI): calcd. form/z C₂₄H₃₂ClFN₁₁O, [M+H]⁺ 526.2553, found 526.2543. HPLC (80% methanolin water): t_(R)=4.211 min, 99.19%.

Example 54

3″-((5-amino-6-chloropyrimidin-4-yl) amino)-2′-fluoro-N,N-dimethyl-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxamideStep 1: Preparation of 1-bromo-2,4-difluoro-3-nitrobenzene

A mixed solution of sulfuric acid (10 mL) and trifluoroacetic acid (50mL) was prepared under an ice bath condition, 2,6-difluoronitrobenzene(5.0 g, 31.4 mmol) was added, and then N-bromosuccinimide (6.15 g, 34.5mmol) was slowly added portionwise. The reaction mixture was stirred atroom temperature for 18 hrs, poured into ice water (100 mL), andextracted with ethyl acetate (3×50 mL). The organic layer was washedwith 2 M sodium hydroxide, dried over anhydrous sodium sulfate, filteredand spin-dried to obtain a red oil (6.6 g). Yield 90.0%; ¹H NMR (300MHz, CDCl₃) δ 7.78 (m, 1H), 7.28-7.16 (m, 1H). m/z (EI-MS): 237.9 [M]⁺

Step 2: Preparation of2′,4′-difluoro-3′-nitro-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 1 of Example 23,4-bromo-1-fluoronitrobenzene was replaced by1-bromo-2,4-difluoro-3-nitrobenzene. Yield 65.0%. ¹H NMR (300 MHz,CDCl₃) δ 7.98-7.96 (m, 2H), 7.91-7.87 (m, 1H), 7.58-7.55 (m, 2H),7.19-7.15 (m, 1H), 3.95 (s, 3H). m/z (EI-MS): 294.1 [M]⁺.

Step 3: Preparation of methyl2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenylyl]-4-carboxylcarboxylate

Following the preparation method in Step 2 of Example 23, methyl4′-fluoro-3′-nitro-[1,1′-biphenylyl]-4-carboxylate was replaced bymethyl 2′,4′-difluoro-3′-nitro-[1,1′-biphenyl]-4-carboxylate. Yield88.2%; m.p. 112° C.-115° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.02-7.99 (m,2H), 7.69-7.62 (m, 3H), 6.98 (d, J=8.6 Hz, 1H), 3.95 (s, 3H), 3.39 (t,J=4.7 Hz, 4H), 2.54 (t, J=4.6 Hz, 4H), 2.34 (s, 3H). m/z (EI-MS): 374.1[M]⁺.

Step 4: Preparation of methyl3′-amino-2′-fluoro-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 3 of Example 23, methyl4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenyl]-4-carboxylate wasreplaced by methyl2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenylyl]-4-carboxylcarboxylate.Yield 73.5%; m.p. 162° C.-165° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.01-7.99(m, 2H), 7.68-7.66 (m, 2H), 6.75 (dd, J=7.5, 5.7 Hz, 1H), 6.58 (d, J=7.5Hz, 1H), 4.35 (s, 2H), 3.95 (s, 3H), 3.39 (t, J=4.7 Hz, 4H), 2.54 (t,J=4.7 Hz, 4H), 2.34 (s, 3H). m z (EI-MS): 344.2 [M]⁺.

Step 5: Preparation of methyl3′-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 4 of Example 23, methyl3′-amino-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate wasreplaced by methyl3′-amino-2′-fluoro-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate.Yield 55.7%; m.p. 222° C.-225° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.11 (s,1H), 8.01-7.99 (m, 2H), 7.80 (s, 1H), 7.62-7.60 (m, 2H), 7.12 (dd,J=7.5, 5.7 Hz, 1H), 6.65 (d, J=7.5 Hz, 1H). 5.35 (s, 2H), 3.95 (s, 3H),3.34 (t, J=4.7 Hz, 4H), 2.57 (t, J=4.7 Hz, 4H), 2.39 (s, 3H). HRMS(ESI): calcd. for m/z C₂₃H₂₄ClFN₆O₂, [M+H]⁺ 471.1706, found 471.1707.HPLC (80% methanol in water): t_(R)=2.744 min, 99.52%.

Step 6: Preparation of 3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylicAcid

Following the preparation method in Step 5 of Example 23, methyl3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate wasreplaced by methyl3′-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate. Yield of two steps: 80.4%; m.p.>250° C.; ¹HNMR (300 MHz, DMSO-d₆) δ 8.11 (dd, J=5.3, 2.2 Hz, 3H), 7.80 (s, 1H),7.69-7.67 (m, 2H), 7.12 (dd, J=7.5, 5.7 Hz, 1H), 6.66 (d, J=7.5 Hz, 1H),5.35 (s, 2H), 3.93 (s, 3H), 3.34 (t, J=4.7 Hz, 4H), 2.57 (s, 4H), 2.32(s, 3H). m/z (EI-MS): 457.2 [M]⁺.

Step 7: Preparation of a Target Product3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-N,N-dimethyl-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxamide

The preparation method was similar to the preparation method in Step 6of Example 23. Yield 77.5%; m.p. 158° C.-160° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 8.03 (s, 1H), 7.71 (s, 1H), 7.60-7.46 (m, 4H), 7.40 (t, J=8.5Hz, 1H), 7.00 (d, J=8.6 Hz, 1H), 5.49 (s, 2H), 2.99-2.98 (m, 6H), 2.87(s, 4H), 2.38 (s, 4H), 2.18 (s, 3H). HRMS (ESI): calcd. for m/zC₂₄H₂₇ClFN₇O, [M+H]⁺ 484.2202, found 484.2203. HPLC (80% methanol inwater): t_(R)=3.448 min, 95.30%.

Example 55

(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(morpholino)methanone

Following the preparation method in Example 54, dimethyl amine wasreplaced by morpholine. Yield 84.7%; m.p. 145° C.-148° C.; ¹H NMR (300MHz, DMSO-d₆) δ 8.05 (s, 1H), 7.72 (s, 1H), 7.54 (dd, J=8.5, 5.4 Hz,4H), 7.41 (t, J=8.5 Hz, 1H), 7.03 (d, J=8.6 Hz, 1H), 5.48 (s, 2H), 3.61(s, 4H), 3.34 (s, 4H), 2.98 (s, 4H), 2.65 (s, 4H), 2.38 (s, 3H). HRMS(ESI): calcd. for m/z C₂₆H₂₉ClFN₇O₂, [M+H]⁺ 526.2128, found 526.2122.HPLC (80% methanol in water): t_(R)=3.560 min, 98.66%.

Example 56

3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-N-(2-morpholinoethyl)-[1,11,1′-terphenyl]-4-carboxamide

Following the preparation method in Example 54, dimethyl amine wasreplaced by 2-morpholinylethyl-1-amine. Yield 74.9%; m.p. 199° C.-202°C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.48 (t, J=5.5 Hz, 1H), 8.09 (s, 1H),7.90 (d, J=8.1 Hz, 2H), 7.71 (s, 1H), 7.59 (d, J=7.9 Hz, 2H), 7.41 (t,J=8.5 Hz, 1H), 7.00 (d, J=8.5 Hz, 1H), 5.50 (s, 2H), 3.57 (s, 4H),3.41-3.38 (m, 2H), 2.91 (s, 4H), 2.49 (s, 4H), 2.42 (s, 6H), 2.21 (s,3H). HRMS (ESI): calcd. for m/z C₂₈H₃₄ClFN₈O₂, [M+H]⁺ 569.2550, found569.2548. HPLC (80% methanol in water): t_(R)=3.634 min, 98.88%.

Example 57

3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-N-(3-morpholinopropyl)-[1,11,1′-terphenyl]-4-carboxamide

Following the preparation method in Example 54, dimethyl amine wasreplaced by 3-morpholinylpropyl-1-amine. Yield 65.2%; m.p. 202° C.-205°C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.46 (t, J=5.7 Hz, 1H), 7.96 (s, 1H),7.81 (d, J=8.2 Hz, 2H), 7.31 (t, J=8.6 Hz, 2H), 6.90 (d, J=8.7 Hz, 1H),5.40 (s, 2H), 3.47 (t, J=5.4 Hz, 4H), 3.21-3.19 (m, 2H), 2.80 (s, 4H),2.28-2.22 (m, 10H), 2.09 (s, 3H), 1.60 (t, J=7.1 Hz, 2H). HRMS (ESI):calcd. for m/z C₂₉H₃₇ClFN₈O₂, [M+H]⁺ 583.2706, found 583.2707. HPLC (80%methanol in water): t_(R)=3.601 min, 97.46%.

Example 58

(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(4-ethylpiperazin-1-yl)methanone

Following the preparation method in Example 54, dimethyl amine wasreplaced by 1-ethylpiperazine. Yield 64.8%; m.p. 229° C.-232° C.; ¹H NMR(300 MHz, DMSO-d₆) δ 8.05 (s, 1H), 7.71 (s, 1H), 7.57 (d, J=8.2 Hz, 2H),7.47 (d, J=8.1 Hz, 2H), 7.39 (t, J=8.5 Hz, 2H), 7.00 (d, J=8.7 Hz, 1H),5.49 (s, 2H), 3.61 (s, 2H), 2.91 (s, 4H), 2.38-2.34 (m, 10H), 2.20 (s,3H), 1.17 (t, J=7.1 Hz, 2H), 1.01 (t, J=7.1 Hz, 2H). HRMS (ESI): calcd.for m/z C₂₈H₃₄ClFN₈O, [M+H]⁺ 553.2587, found 553.2596. HPLC (80%methanol in water): t_(R)=3.794 min, 97.08%.

Example 59

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)tetrahydro-2H-pyran-4-carboxamideStep 1: Preparation of 2′,4′-difluoro-3′-nitro[1,1′-biphenyl]-4-amine

Following the preparation method in Step 1 of Example 23,4-bromo-1-fluoronitrobenzene was replaced by1-bromo-2,4-difluoro-3-nitrobenzene. Yield 69.0%; m.p. 136° C.-138° C.;¹H NMR (300 MHz, DMSO-d₆) δ 7.87-7.83 (m, 1H), 7.24-7.22 (m, 2H), 7.15(dd, J=9.0, 7.5 Hz, 1H), 6.72-6.70 (m, 2H), 5.24 (s, 2H). m/z (EI-MS):251.1 [M]⁺.

Step 2: Preparation of2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro[1,1′-biphenyl]-4-amine

Following the preparation method in Step 2 of Example 23, methyl4′-fluoro-3′-nitro-[1,1′-biphenylyl]-4-carboxylate was replaced by2′,4′-difluoro-3′-nitro[1,1′-biphenyl]-4-amine. Yield 88.2%; m.p. 145°C.-147° C.; ¹H NMR (300 MHz, DMSO-d₆) δ δ 7.60 (dd, J=7.4, 5.8 Hz, 1H),7.25-7.18 (m, 2H), 6.96 (d, J=7.5 Hz, 1H), 6.77-6.71 (m, 2H), 5.24 (s,2H), 3.20 (t, J=4.6 Hz, 4H), 2.54 (t, J=4.6 Hz, 4H), 2.34 (s, 3H). m/z(EI-MS): 331.2 [M]⁺.

Step 3: Preparation ofN-(2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro[1,1′-biphenyl]-4-yl)tetrahydro-2H-pyran-4-carboxamide

Following a preparation method similar to that in Example 33,N-(4′-amino-4-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-3-yl)-6-chloropyrimidine-e-4,5-diamine was replaced by2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro[1,1′-biphenyl]-4-amine.Yield 88.2%; m.p. 166° C.-168° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.52 (s,1H), 7.63 (dd, J=7.5, 5.7 Hz, 1H), 7.42-7.38 (m, 4H), 6.99 (d, J=7.5 Hz,1H), 3.95-3.92 (m, 2H), 3.53-3.50 (m, 2H), 3.20 (t, J=4.7 Hz, 4H), 2.54(t, J=4.7 Hz, 4H), 2.65-2.60 (m, 4H), 2.08-2.01 (m, 2H), 1.83-1.76 (m,2H). m/z (EI-MS): 443.2 [M]⁺.

Step 4: Preparation of a Target ProductN-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)tetrahydro-2H-pyran-4-carboxamide

Following a preparation method similar to that in Step 3 and Step 4 ofExample 23, a compound 59 was obtained. Yield of three steps: 36.6%;m.p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.56 (s, 1H), 8.25 (s, 1H),7.80-7.77 (m, 3H), 7.41-7.39 (m, 2H), 7.07 (dd, J=7.5, 5.7 Hz, 1H), 6.68(d, J=7.3 Hz, 1H), 5.43 (s, 2H), 3.52-3.49 (m, 4H), 3.34 (t, J=4.6 Hz,4H), 2.70-2.67 (m, 1H), 2.44 (s, 4H), 2.22 (s, 3H), 2.06-1.99 (m, 2H),1.85-1.78 (m, 2H). HRMS (ESI): calcd. for m/z C₂₇H₃₁ClFN₇O₂, [M+H]⁺540.2285, found 540.2276. HPLC (90% methanol in water): t_(R)=3.656 min,98.94%.

Example 60

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-pyridin-4-yl)-1-methylpiperidin-4-carboxamideStep 1: Preparation of N-(2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro[1,1′-biphenyl]-4-yl)-1-methylpiperidin-4-carboxamide

Following the preparation method in Step 3 of Example 59,tetrahydropyran-4-carboxylic acid was replaced by1-methylpiperazine-4-carboxylic acid. Yield 75.3%; m.p. 170° C.-172° C.;¹H NMR (300 MHz, DMSO-d₆) δ 9.55 (s, 1H), 7.63 (dd, J=7.5, 5.7 Hz, 1H),7.40 (s, 4H), 6.98 (d, J=7.5 Hz, 1H), 3.22 (t, J=4.7 Hz, 4H), 2.99-2.93(m, 6H), 2.64-2.58 (m, 4H), 2.37 (s, 3H), 2.29-2.24 (m, 2H), 2.09-2.02(m, 2H), 1.71-1.64 (m, 2H). m/z (EI-MS): 455.2 [M]⁺.

Step 2: Preparation of a Target ProductN-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-pyridin-4-yl)-1-methylpiperidin-4-carboxamide

Following a preparation method similar to that in Step 3 and Step 4 ofExample 23, a compound 60 was obtained. Yield of three steps: 35.3%;m.p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.18 (s, 1H),7.80 (s, 1H), 7.63 (d, J=7.5, 2H), 7.40 (d, J=7.6, 2H), 7.08-7.05 (m,1H), 6.61 (d, J=7.5 Hz, 1H), 5.30 (s, 2H), 3.20 (t, J=4.9 Hz, 4H),3.01-2.96 (m, 6H), 2.63-2.60 (m, 4H), 2.37 (s, 3H), 2.21-2.18 (m, 4H),2.07-2.02 (m, 4H). HRMS (ESI): calcd. for m/z C₂₈H₃₄ClFN₈O, [M+H]⁺553.2607, found 553.2606. HPLC (80% methanol in water): t_(R)=3.168 min,97.33%.

Example 61

N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-piperidin-4-yl)-2-(tetrahydro-2H-pyran-4-yl)acetamideStep 1: Preparation ofN-(2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro[1,1′-biphenyl]-4-yl)-2-(tetrahydro-2H-pyran-4-yl)acetamide

Following Step 3 of Example 59, tetrahydropyran-4-carboxylic acid wasreplaced by tetrahydropyran-4-ethanoic acid. Yield 68.9%; m.p. 148°C.-150° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 7.80-7.77 (m, 2H), 7.70 (s, 1H),7.65-7.63 (m, 1H), 7.39 (d, J=7.5 Hz, 2H), 6.94 (d, J=7.5 Hz, 1H),3.90-3.87 (m, 2H), 3.55-3.51 (m, 2H), 3.24 (t, J=4.7 Hz, 4H), 2.60 (s,3H), 2.38 (t, J=4.7 Hz, 4H), 2.15 (d, J=7.0 Hz, 2H), 2.05-1.99 (m, 1H),1.20-1.17 (m, 4H). m/z (EI-MS): 457.2 [M]⁺.

Step 2: Preparation of a Target ProductN-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-piperidin-4-yl)-2-(tetrahydro-2H-pyran-4-yl)acetamide

Following a preparation method similar to that in Step 3 and Step 4 ofExample 23, a compound 61 was obtained. Yield of three steps: 38.6%;m.p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.24 (s, 1H), 8.11 (s, 1H),7.80 (s, 1H), 7.40 (s, 4H), 7.15-7.12 (m, 1H), 6.73 (d, J=7.5 Hz, 1H),5.42 (s, 2H), 3.55-3.50 (m, 4H), 3.53 (t, J=4.6 Hz, 4H), 2.48 (s, 4H),2.26 (s, 3H), 2.15 (d, J=7.0 Hz, 2H), 2.03-2.01 (m, 1H), 1.70-1.64 (m,4H). HRMS (ESI): calcd. for m/z C₂₈H₃₃ClFN₇O₂, [M+H]⁺ 554.2441, found554.2440. HPLC (80% methanol in water): t_(R)=3.524 min, 99.10%.

Example 62

2-amino-N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′biphenylyl]-4-yl)-3-methylbutyramide Step 1: Preparation of(1-((2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenyl]-4-yl)amino)-3-methyl-t-butyl-1-oxobutan-2-yl)aminocarboxylicAcid

Following Step 3 of Example 59, tetrahydropyran-4-carboxylic acid wasreplaced by (t-butoxylcarbonyl)valine. Yield 69.0%; m.p. 108° C.-110°C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.63 (s, 1H), 7.64-7.61 (m, 3H),7.44-7.42 (m, 2H), 7.39 (s, 3H), 6.98 (d, J=7.5 Hz, 1H), 4.13-4.12 (m,1H), 3.75-3.72 (m, 2H), 3.31-3.27 (m, 2H), 2.79-2.77 (m, 2H), 2.63-2.61(m, 1H), 2.60 (s, 3H), 2.24-2.22 (m, 2H), 1.44 (s, 9H), 0.94-0.88 (m,6H). m/z (EI-MS): 530.3 [M]⁺.

Step 2: Preparation of a Target Product2-amino-N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′biphenylyl]-4-yl)-3-methylbutyramide

Following a preparation method similar to that in Step 3 and Step 4 ofExample 23, a compound 62 was obtained. Yield of three steps: 46.9%,m.p. 189° C.-192° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.00 (s, 1H),7.74-7.71 (m, 3H), 7.45 (s, 2H), 7.33 (t, J=8.5 Hz, 1H), 6.96 (d, J=7.2Hz, 1H), 5.47 (s, 2H), 3.15-3.12 (m, 1H), 2.86 (s, 4H), 2.36 (s, 4H),2.16 (s, 3H), 1.95 (s, 2H), 1.17 (s, 1H), 0.93 (s, 3H), 0.86 (s, 3H).HRMS (ESI): calcd. for m/z C₂₆H₃₂ClFN₈O, [M+H]⁺ 527.2444, found527.2449. HPLC (80% methanol in water): t_(R)=3.858 min, 99.62%.

Example 63

2-amino-N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′biphenylyl]-4-yl)-4-methylvaleramide Step 1:Preparation of (1-((2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenyl]-4-yl)amino)-4-methyl-t-butyl-1-oxopentan-2-yl)aminocarboxylicAcid

Following Step 3 of Example 59, tetrahydropyran-4-carboxylic acid wasreplaced by (t-butoxylcarbonyl) leucine. Yield 67.2%; m.p. 114° C.-116°C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.46 (s, 1H), 7.65-7.62 (m, 1H),7.45-7.42 (m, 4H), 6.95 (d, J=7.5 Hz, 1H), 6.24 (s, 1H), 4.04-4.01 (m,1H), 3.87-3.82 (m, 2H), 3.31-3.25 (m, 2H), 2.79-2.74 (m, 2H), 2.25-2.19(m, 4H), 1.98-1.92 (m, 2H), 1.71-1.65 (m, 1H), 1.44 (s, 9H), 0.96-0.90(m, 6H). m/z (EI-MS): 544.3 [M]⁺.

Step 2: Preparation of a Target Product2-amino-N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-yl)-4-methylvaleramide

Following a preparation method similar to that in Step 3 and Step 4 ofExample 23, a compound 63 was obtained. Yield of three steps: 47.6%;m.p. 182° C.-185° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 7.99 (s, 1H),7.73-7.71 (m, 3H), 7.45 (d, J=8.1 Hz, 2H), 7.33 (t, J=8.5 Hz, 2H), 6.96(d, J=8.6 Hz, 2H), 5.45 (s, 2H), 3.38-3.36 (m, 1H), 2.87 (s, 4H), 2.36(s, 4H), 2.17 (s, 3H), 1.78-1.74 (m, 2H), 1.52-1.47 (m, 2H), 1.19-1.17(m, 1H), 0.90 (t, J=7.2 Hz, 6H). m/z (EI-MS): 541.3 [M]⁺. HPLC (80%methanol in water): t_(R)=3.680 min, 98.32%.

Example 64

2-amino-N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′biphenylyl]-4-yl)acetamideStep 1: Preparation of(2-((2′-fluoro-4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenyl]-4-yl)amino)-2-oxoethyl-t-butyl)aminocarboxylate

Following Step 3 of Example 59, tetrahydropyran-4-carboxylic acid wasreplaced by (t-butoxylcarbonyl)glycine. Yield 70.0%; m.p. 112° C.-114°C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.20 (s, 1H), 7.63 (dd, J=7.5, 5.7 Hz,1H), 7.49-7.47 (m, 2H), 7.44-7.42 (m, 2H), 6.94 (d, J=7.5 Hz, 1H), 6.31(s, 1H), 3.89 (s, 2H), 3.30 (t, J=4.7 Hz, 4H), 2.44 (s, 4H), 2.24 (s,3H), 1.44 (s, 9H). m/z (EI-MS): 486.2 [M]⁺.

Step 2: Preparation of a Target Product2-amino-N-(3″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′biphenylyl]-4-yl)acetamide

Following a preparation method similar to that in Step 3 and Step 4 ofExample 23, a compound 64 was obtained. Yield of three steps: 50.9%;m.p. 177° C.-179° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 7.88 (s, 1H),7.63-7.61 (m, 3H), 7.36 (d, J=8.2 Hz, 2H), 7.24 (t, J=8.5 Hz, 2H), 6.87(d, J=8.5 Hz, 2H), 5.35 (s, 2H), 4.00 (s, 1H), 3.08 (s, 2H), 2.77 (t,J=4.7 Hz, 4H), 2.27 (t, J=4.6 Hz, 4H), 2.07 (s, 3H). m/z (EI-MS): 485.3[M]⁺. HPLC (80% methanol in water): t_(R)=3.595 min, 99.76%.

Example 65

3″-((5-amino-6-chloropyrimidin-4-yl)amino)-N,N,2′-trimethyl-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxamideStep 1: Preparation of 1-bromo-4-fluoro-2-methyl-3-nitrobenzene

Following the preparation method in Step 1 of Example 54,2,6-difluoronitrobenzene was replaced by1-bromo-2,4-difluoro-3-nitrobenzene. Yield 86.7%; m.p. 74° C.-77° C.; ¹HNMR (300 MHz, CDCl₃) δ 7.72 (d, J=7.5, Hz, 1H), 7.03 (d, J=7.5 Hz, 1H),2.32 (s, 3H). m/z (EI-MS): 233.9 [M]⁺

Step 2: Preparation of4′-fluoro-N,N,2′-trimethyl-3′-nitro-[11,1′-biphenyl]-4-carboxamide

Following the preparation method in Step 2 of Example 54,(4-(methoxylcarbonyl)phenyl)boric acid was replaced by(4-(dimethylaminoformoxyl)phenyl)boric acid. Yield 76.9%; ¹H NMR (300MHz, CDCl₃) δ 7.89-7.86 (m, 2H), 7.76 (d, J=7.5, Hz, 1H), 7.51-7.49 (m,2H), 7.26 (d, J=7.5 Hz, 1H), 3.03 (s, 6H), 2.30 (s, 3H). m/z (EI-MS):303.1 [M]⁺.

Step 3: Preparation ofN,N,2′-trimethyl-4′-(4-methylpiperazin-1-yl)-3′-nitro[1,1′-biphenyl]-4-carboxamide

Following the preparation method in Step 3 of Example 54, methyl4′-fluoro-3′-nitro-[1,1′-biphenylyl]-4-carboxylate was replaced by4′-fluoro-N,N,2′-trimethyl-3′-nitro-[1,1′-biphenyl]-4-carboxamide. Yield85.5%; m.p. 110° C.-112° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 7.91 (d, J=5.7Hz, 2H), 7.60-7.57 (m, 3H), 7.04 (d, J=5.6 Hz, 1H), 3.39 (t, J=4.7 Hz,4H), 3.03 (s, 6H), 2.54 (t, J=4.6 Hz, 4H), 2.34 (s, 3H), 2.17 (s, 3H).m/z (EI-MS): 383.2 [M]⁺.

Step 4: Preparation of3′-amino-N,N,2′-trimethyl-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxamide

Following the preparation method in Step 4 of Example 54, methyl4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenyl]-4-carboxylate wasreplaced by N,N,2′-trimethyl-4′-(4-methylpiperazin-1-yl)-3′-nitro[1,1′-biphenyl]-4-carboxamide.Yield 80.5%; m.p. 158° C.-160° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 7.91-7.89(m, 2H), 7.61-7.59 (m, 2H), 6.68-6.64 (m, 2H), 4.35 (s, 2H), 3.39 (t,J=4.7 Hz, 4H), 3.03 (s, 6H), 2.54 (s, 4H), 2.14 (s, 3H), 2.09 (s, 3H).m/z (EI-MS): 353.2 [M]⁺.

Step 5: Preparation of a Target Product3″-((5-amino-6-chloropyrimidin-4-yl)amino)-N,N,2′-trimethyl-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxamide

Following the preparation method in Step 5 of Example 54, a compound 65was obtained. Yield 44.5%; m.p. 165° C.-167° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 8.09 (s, 1H), 7.87-7.85 (m, 2H), 7.80 (s, 1H), 7.58 (d, J=7.4Hz, 2H), 7.10 (d, J=7.5 Hz, 1H), 6.86 (d, J=7.5 Hz, 1H), 5.49 (s, 2H),3.01-2.98 (m, 6H), 2.87 (s, 4H), 2.38 (s, 4H), 2.18 (s, 3H), 2.09 (s,3H). m/z (EI-MS): 480.2 [M]⁺. HPLC (80% methanol in water): t_(R)=3.543min, 96.37%.

Example 66

Methyl5′-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate Step 1: Preparation of methyl2′,4′-difluoro-5′-nitro-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 1 of Example 23,4-bromo-1-fluoronitrobenzene was replaced by1-bromo-2,4-difluoro-5-nitrobenzene. Yield 67.8%; m.p. 134° C.-136° C.;¹H NMR (300 MHz, CDCl₃) δ 8.30 (t, J=5.7 Hz, 1H), 7.97 (d, J=8.9 Hz,2H), 7.57 (d, J=5.8 Hz, 2H), 7.24 (t, J=8.9 Hz, 1H), 3.95 (s, 3H). m/z(EI-MS): 294.1 [M]⁺.

Step 2: Preparation of methyl2′-fluoro-4′-(4-methylpiperazin-1-yl)-5′-nitro-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 2 of Example 23, methyl4′-fluoro-3′-nitro-[1,1′-biphenylyl]-4-carboxylate was replaced bymethyl 2′,4′-difluoro-5′-nitro-[1,1′-biphenyl]-4-carboxylate. Yield84.3%; m.p. 137° C.-140° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.02-7.99 (m,3H), 7.68-7.65 (m, 2H), 6.79 (d, J=7.5 Hz, 1H), 3.95 (s, 3H), 3.39 (t,J=4.7 Hz, 4H), 2.54 (t, J=4.6 Hz, 4H), 2.14 (s, 3H). m/z (EI-MS): 374.1[M]⁺.

Step 3: Preparation of methyl5′-amino-2′-fluoro-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 3 of Example 23, methyl4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenyl]-4-carboxylate wasreplaced by methyl2′-fluoro-4′-(4-methylpiperazin-1-yl)-5′-nitro-[1,1′-biphenyl]-4-carboxylate.Yield 80.6%; m.p. 162° C.-165° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.00 (d,J=8.9 Hz, 2H), 7.68 (d, J=8.8 Hz, 2H), 6.56 (d, J=5.7 Hz, 1H), 6.39 (d,J=8.9 Hz, 1H), 4.35 (s, 2H), 4.35 (s, 2H), 3.95 (s, 3H), 3.39 (t, J=4.7Hz, 4H), 2.54 (t, J=4.7 Hz, 4H), 2.17 (s, 3H). m/z (EI-MS): 344.2 [M]⁺.

Step 4: Preparation of a Target Product methyl5′-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate

Following a preparation method similar to that in Step 4 of Example 23,a compound 66 was obtained. Yield 57.4%; m.p. 220° C.-223° C.; ¹H NMR(300 MHz, DMSO-d₆) δ 8.14 (s, 1H), 8.02-7.99 (m, 2H), 7.80 (s, 1H),7.66-7.64 (m, 2H), 6.80 (d, J=5.7 Hz, 1H), 6.56 (d, J=8.9 Hz, 1H), 5.35(s, 2H), 3.95 (s, 3H), 3.34 (t, J=4.7 Hz, 4H), 2.57 (t, J=4.7 Hz, 4H),2.19 (s, 3H). HRMS (ESI): calcd. for m/z C₂₃H₂₄ClFN₆O₂, [M+H]⁺ 471.1706,found 471.1706. HPLC (80% methanol in water): t_(R)=4.248 min, 98.83%.

Example 67

(5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(morpholino)methanoneStep 1: Preparation of5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylicAcid

Following the preparation method in Step 5 of Example 23, methyl3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate was replaced by methyl5′-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate. Yield 82.7%; m.p.>250° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 8.14-8.10 (m, 3H), 7.80 (s, 1H), 7.73-7.70 (m, 2H), 6.80 (d,J=5.7 Hz, 1H), 6.57 (d, J=8.9 Hz, 1H), 5.34 (s, 2H), 3.93 (s, 3H), 3.34(t, J=4.7 Hz, 4H), 2.57 (s, 4H), 2.12 (s, 3H). m/z (EI-MS): 457.2 [M]⁺.

Step 2: Preparation of a Target Product(5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)(morpholino)methanone

Following a preparation method similar to that in Step 6 of Example 23,a target compound 67 was obtained. Yield 78.4%; m.p. 136° C.-138° C.; ¹HNMR (300 MHz, DMSO-d₆) δ 8.09 (s, 1H), 7.93 (d, J=8.7 Hz, 1H), 7.82 (s,1H), 7.59 (s, 2H), 7.51 (d, J=7.8 Hz, 2H), 7.12 (d, J=8.6 Hz, 1H), 5.40(s, 2H), 3.61 (s, 4H), 2.97 (s, 4H), 2.72 (s, 4H), 2.54-2.51 (m, 4H),2.41 (s, 3H). HRMS (ESI): calcd. for m/z C₂₆H₂₉ClFN₇O₂, [M+H]⁺ 526.2128,found 526.2130. HPLC (80% methanol in water): t_(R)=4.046 min, 96.85%.

Example 68

5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-N-(2-morpholinoethyl)-[1,11,1′-terphenyl]-4-carboxamide

Following a preparation method similar to that in Example 67, morpholinewas replaced by 1-ethylmorpholine, to obtain a target compound 68. Yield78.4%; m.p. 199° C.-202° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.47 (s, 1H),8.09 (s, 1H), 7.95-7.92 (m, 3H), 7.81 (s, 1H), 7.62 (d, J=7.9 Hz, 2H),7.11 (d, J=12.4 Hz, 1H), 5.39 (s, 2H), 3.58 (t, J=4.6 Hz, 4H), 3.41-3.40(m, 4H), 2.92 (s, 4H), 2.56 (s, 4H), 2.45 (s, 4H), 2.29 (s, 3H). HRMS(ESI): calcd. for m/z C₂₈H₃₄ClFN₈O₂, [M+H]⁺ 569.2550, found 569.2548.HPLC (80% methanol in water): t_(R)=4.049 min, 98.64%.

Example 69

5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-N-(3-morpholinopropyl)-[1,11,1′-terphenyl]-4-carboxamide

Following a preparation method similar to that in Example 67, morpholinewas replaced by 1-propylmorpholine, to obtain a target product 69. Yield78.4%; m.p. 204° C.-206° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.55 (s, 1H),8.09 (s, 1H), 7.93-7.90 (m, 3H), 7.81 (s, 1H), 7.63-7.60 (m, 2H), 7.11(d, J=12.3 Hz, 1H), 5.39 (s, 2H), 3.59 (s, 4H), 3.32 (s, 4H), 2.92 (s,4H), 2.58 (s, 4H), 2.41 (s, 4H), 2.31 (s, 3H), 1.72 (s, 2H). HRMS (ESI):calcd. for m/z C₂₉H₃₆ClFN₈O₂, [M+H]⁺ 583.2693, found 583.2699. HPLC (80%methanol in water): t_(R)=3.885 min, 99.38%.

Example 70

5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-N-(4-hydroxylcyclohexyl)-4″-(4-methylpiperazin-1-yl)-[1,11,1′-terphenyl]-4-carboxamide

Following a preparation method similar to that in Example 67, morpholinewas replaced by piperidin-4-ol, to obtain a target compound 70. Yield65.2%; m.p. 200° C.-203° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 9.67 (s, 1H),8.06 (s, 1H), 7.92 (d, J=8.4 Hz, 1H), 7.82 (s, 1H), 7.58 (d, J=7.7 Hz,2H), 7.47 (d, J=7.9 Hz, 2H), 7.21 (d, J=12.2 Hz, 1H), 5.44 (s, 2H), 4.82(s, 1H), 4.01 (s, 2H), 3.52 (s, 2H), 3.16 (s, 8H), 2.78 (s, 3H), 1.76(s, 2H), 1.36 (s, 2H). HRMS (ESI): calcd. for m/z C₂₇H₃₁ClFN₇O₂, [M+H]⁺554.2441, found 554.2442. HPLC (80% methanol in water): t_(R)=4.057 min,98.48%.

Example 71

5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-4″-(4-methylpiperazin-1-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)-[1,1′-biphenylyl]-4-carboxamide

Following a preparation method similar to that in Example 67, morpholinewas replaced by (tetrahydro-2H-pyran-4-yl)methylamine, to obtain atarget compound 71. Yield 75.2%; m.p. 189° C.-191° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 8.54 (s, 1H), 8.10 (s, 1H), 7.94-7.91 (m, 3H), 7.61 (d, J=8.0Hz, 2H), 7.11 (d, J=12.2 Hz, 1H), 5.40 (s, 2H), 3.87-3.83 (m, 2H), 3.17(s, 4H), 2.92 (s, 4H), 2.57 (s, 4H), 2.30 (s, 3H), 1.81 (s, 1H), 1.23(s, 4H). HRMS (ESI): calcd. for m/z C₂₈H₃₃ClFN₇O₂, [M+H]⁺ 540.2285,found 540.2284. HPLC (100% methanol): t_(R)=7.989 min, 99.16%.

Example 72

5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-fluoro-N,N-dimethyl-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxamide

Following a preparation method similar to that in Example 67, morpholinewas replaced by dimethyl amine, to obtain a target compound 72. Yield75.2%; m.p. 157° C.-159° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.01 (s, 1H),7.83 (dd, J=8.7, 3.7 Hz, 1H), 7.72 (d, J=3.6 Hz, 1H), 7.49 (d, J=7.1 Hz,2H), 7.45-7.36 (m, 2H), 7.14-7.01 (m, 1H), 5.35 (s, 2H), 3.27-2.87 (m,14H), 2.40 (s, 3H). HRMS (ESI): calcd. for m/z C₂₄H₂₇ClFN₇O, [M+H]⁺484.2022, found 484.2022. HPLC (80% methanol in water): t_(R)=4.751 min,95.34%.

Example 73

Methyl 5′-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-methyl-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylateStep 1: Preparation of methyl4′-fluoro-2′-methyl-5′-nitro-[11,1′-biphenylyl]-4-carboxylate

Following the preparation method in Step 1 of Example 23,4-bromo-1-fluoronitrobenzene was replaced by1-bromo-4-fluoro-2-methyl-5-nitrobenzene. Yield 68.9%; m.p. 133° C.-134°C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.06 (dd, J=9.4, 3.2 Hz, 2H), 7.97 (d,J=7.7 Hz, 1H), 7.62-7.57 (m, 3H), 3.89 (s, 3H), 2.33 (s, 3H). m/z(EI-MS): 290.1 [M]⁺.

Step 2: Preparation of methyl2′-methyl-4′-(4-methylpiperazin-1-yl)-5′-nitro-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 2 of Example 23, methyl4′-fluoro-3′-nitro-[1,1′-biphenylyl]-4-carboxylate was replaced bymethyl 4′-fluoro-2′-methyl-5′-nitro-[1,1′-biphenylyl]-4-carboxylate.Yield 84.3%; m.p. 153° C.-155° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.02-7.99(m, 2H), 7.84 (s, 1H), 7.61-7.58 (m, 2H), 7.09 (s, 1H), 3.95 (s, 3H),3.39 (t, J=4.7 Hz, 4H), 2.54 (t, J=4.6 Hz, 4H), 2.32 (d, J=1.0 Hz, 3H),2.14 (s, 3H). m/z (EI-MS): 370.1 [M]⁺.

Step 3: Preparation of methyl5′-amino-2′-methyl-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 3 of Example 23, methyl4′-(4-methylpiperazin-1-yl)-3′-nitro-[1,1′-biphenyl]-4-carboxylate wasreplaced by methyl2′-methyl-4′-(4-methylpiperazin-1-yl)-5′-nitro-[1,1′-biphenyl]-4-carboxylate.Yield 78.5%; m.p. 158° C.-160° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.00 (d,J=8.9 Hz, 2H), 7.68 (d, J=8.8 Hz, 2H), 6.56 (d, J=5.7 Hz, 1H), 6.39 (d,J=8.9 Hz, 1H), 4.35 (s, 2H), 4.35 (s, 2H), 3.95 (s, 3H), 3.39 (t, J=4.7Hz, 4H), 2.54 (t, J=4.7 Hz, 4H), 2.17 (s, 3H). m/z (EI-MS): 340.2 [M]⁺.

Step 4: Preparation of a target compound methyl5′-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-methyl-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate

Following the preparation method in Step 4 of Example 23, methyl3′-amino-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate wasreplaced by methyl5′-amino-2′-methyl-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate.Yield 45.9%; m.p. 225° C.-228° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.14 (s,1H), 8.00 (d, J=7.7 Hz, 2H), 7.80 (d, J=4.4 Hz, 2H), 7.50-7.47 (m, 2H),7.08 (s, 1H), 5.30 (s, 2H), 2.86 (d, J=5.5 Hz, 4H), 2.47 (s, 4H), 2.22(d, J=8.0 Hz, 6H). HRMS (ESI): calcd. for m/z C₂₄H₂₇ClN₆O₂, [M+H]⁺467.1957, found 467.1953. HPLC (80% methanol in water): t_(R)=3.844 min,97.34%.

Example 74

5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-methyl-4″-(4-methylpiperazin-1-yl)-N-(3-morpholinopropyl)-[1,11,1′-terphenyl]-4-carboxamideStep 1: Preparation of5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-methyl-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylicAcid

Following the preparation method in Step 5 of Example 23, methyl3′-((5-amino-6-chloropyrimidin-4-yl)amino)-4′-(4-methylpiperazin-1-yl)-[1,1′-biphenylyl]-4-carboxylate was replaced by methyl5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-methyl-4″-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-carboxylate.Yield 79.4%; m.p.>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 8.13-8.10 (m,3H), 7.80 (s, 1H), 7.66-7.62 (m, 2H), 6.85 (s, 1H), 6.77 (s, 1H), 5.30(s, 2H), 2.98 (d, J=5.1 Hz, 4H), 2.47 (s, 4H), 2.24 (d, J=8.0 Hz, 6H). mz (EI-MS): 453.2 [M]⁺.

Step 2: Preparation of a target compound5″-((5-amino-6-chloropyrimidin-4-yl)amino)-2′-methyl-4″-(4-methylpiperazin-1-yl)-N-(3-morpholinopropyl)-[1,11,1′-terphenyl]-4-carboxamide

Following a preparation method similar to that in Example 67, a compound74 was obtained. Yield 78.4%; m.p. 204° C.-206° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 8.65 (s, 1H), 8.10 (s, 1H), 7.93-7.90 (m, 3H), 7.80 (s, 1H),7.66-7.62 (m, 2H), 7.11 (d, J=12.3 Hz, 1H), 5.39 (s, 2H), 3.59 (s, 4H),3.32 (s, 4H), 2.92 (s, 4H), 2.58 (s, 4H), 2.41 (s, 4H), 2.31 (s, 6H),1.72 (s, 2H). HRMS (ESI): calcd. for m/z C₂₀H₃₉ClN₈O₂, [M+H]⁺ 579.2944,found 579.2950. HPLC (80% methanol in water): t_(R)=3.830 min, 98.82%.

Test Example 1: Some Pharmacodynamic Tests and Results

In the present disclosure, a fluorescent molecular probe was constructedbased on a MLL1 peptide fragment binding to WDR5, for use as a methodfor study on an aniline compound interfering with WDR5 protein-proteininteraction, to determine an inhibition rate of the aniline compound atvarious concentrations, and then compute the IC₅₀ value. Specificexperimental steps: 20 μL of WDR5 protein, 20 μL of a fluorescent probe,and 20 μL of a compound of different concentration gradients were addedto a 384-well plate respectively. After incubation for 0.5 hrs, thefluorescence was read on a multifunctional microplate reader at anexcitation wavelength of 485 nm and an emission wavelength of 535 nm, tocompute the mP value. The inhibition rate was computed as per theformula below, and then the IC₅₀ value was computed using GraphPadsoftware. The results are shown in Table 1.

${{Inhibition}\mspace{14mu}{rate}} = {\frac{\begin{matrix}{{{mP}\mspace{14mu}{value}\mspace{14mu}{of}\mspace{14mu}{compound}\mspace{14mu}{group}} -} \\{{mP}\mspace{14mu}{value}{\mspace{11mu}\;}{of}\mspace{14mu}{blank}\mspace{14mu}{group}}\end{matrix}}{\begin{matrix}{{{mP}\mspace{14mu}{value}\mspace{14mu}{of}\mspace{14mu}{negative}\mspace{14mu}{control}\mspace{14mu}{group}} -} \\{{mP}\mspace{14mu}{value}\mspace{14mu}{of}\mspace{14mu}{blank}\mspace{14mu}{group}}\end{matrix}} \times 100\%}$

Interfering with the WDR5 protein-protein interaction will affect theH3K4 methyltransferase activity of MLL1, thereby down-regulating theexpression of Hox and Meis-1 genes and inhibiting the proliferation ofleukemia cells.

A biphenyl compound DDO-2084 is a micromolecular inhibitor that has beenreported to be capable of inhibiting WDR5 protein-protein interaction(Eur. J. Med. Chem. 2016, 124, 480-489.). In the present disclosure,DDO-2084 is used as a positive control compound.

TABLE 1 Activity of the Compound of the Present Disclosure in InhibitingWDR5 Protein-Protein Interaction and Methyltransferase Activity Whetherthe MLL1-WDR5 methylation Example PPI inhibiting level of compoundactivity H3K4 was No^(a) IC₅₀ (μM) inhibited  2 10.3 ND^(c)  7 2.66ND^(c) 23 0.186 Yes 24 0.183 Yes 25 0.165 Yes 26 0.36 Yes 27 0.217 Yes28 0.270 Yes 29 0.370 Yes 30 0.310 Yes 31 0.46 Yes 32 0.691 Yes 33 0.489Yes 34 0.287 Yes 35 0.372 Yes 36 0.261 Yes 37 5.55 Yes 38 0.652 Yes 390.675 Yes 40 0.328 Yes 41 0.329 Yes 42 1.56 ND^(c) 43 11.43 ND^(c) 445.707 ND^(c) 45 0.56 Yes 46 0.865 Yes 47 0.24 Yes 48 1.09 ND^(c) 49 1.25ND^(c) 50 0.164 Yes 51 0.86 Yes 52 1.56 ND^(c) 53 1.61 ND^(c) 54 0.24Yes 55 0.117 Yes 56 0.102 Yes 57 0.069 Yes 58 0.112 Yes 59 0.176 Yes 600.105 Yes 61 0.085 Yes 62 0.034 Yes 63 0.025 Yes 64 0.033 Yes 65 0.671ND^(c) 66 0.040 Yes 67 0.036 Yes 68 0.074 Yes 69 0.021 Yes 70 0.033 Yes71 0.036 Yes 72 0.029 Yes 73 0.316 Yes 74 0.465 Yes DDO-2084^(b) 338.2Yes ^(a)The structure of the compound is as shown in specific examples;^(b)the structure of DDO-2084:

^(c)ND: not detected.

As can be seen from Table 1, the compound of the present disclosure hasa potent activity in inhibiting WDR5 protein-protein interaction. Table1 shows the experimental results regarding whether some of the compoundsof the present disclosure have the effect of inhibiting the methylationlevel of H3K4. The experimental results show that the compounds of thepresent disclosure that have an inhibitory effect on the WDR5protein-protein interaction can all down-regulate the methylation levelof H3K4.

Some compounds of the present disclosure have also been tested for theantiproliferative activity against leukemia cells. Table 2 shows theresults for evaluating the activity of some compounds of the presentdisclosure in inhibiting the proliferation of acute leukemia cells, inwhich MV4-11 is human acute mononuclear leukemia cells, and Molm-13 ishuman acute myeloid leukemia cells. Table 2 shows that the compounds ofthe present disclosure have the effect of significantly inhibiting theproliferation of various leukemia cells.

TABLE 2 Anti-proliferative Activity of Some Compounds of the PresentDisclosure against Leukemia Cells Example Example compound GI₅₀/μM GI₅₀/compound GI₅₀/ GI₅₀/ No^(a) (MV-411) μM(Molm-13) No^(a) μM(MV-411)μM(Molm-13) 23 16.67 23.41 50 29.61 35.48 24 11.00 17.64 51 37.95 38.9525 24.90 20.83 54 17.48 16.16 26 48.62 30.73 55 22.49 28.87 27 39.2433.96 56 9.037 8.43 28 29.67 24.31 57 22.28 17.62 29 16.56 18.17 5819.47 20.30 30 27.29 32.64 59 26.43 31.23 31 10.50 18.78 60 22.73 22.6032 40.01 27.19 61 21.05 20.53 33 25.86 22.72 62 17.40 21.98 34 23.2020.76 63 19.65 19.36 35 2.07 6.164 64 20.45 28.64 36 9.32 12.49 66 14.39ND 38 20.84 26.01 67 19.56 16.50 39 32.20 28.62 68 27.70 23.10 40 28.3926.25 69 25.64 17.27 41 25.80 24.40 70 20.17 13.16 45 27.27 23.17 7115.73 12.29 46 22.35 11.68 72 12.57 13.09 47 10.61 14.70 DDO-2084 17.7 ±2.3 ND^(b) ^(a)The structure of the compound is as shown in specificexamples; ^(b)ND: not detected;

Moreover, some compounds have also been tested by Western-blot. Theresults of the inhibitory effect on the methyltransferase function ofMLL1 at the cellular level are shown in FIG. 1. As can be seen from FIG.1, the title compound of Example 72 can inhibit the catalytic activityof MLL1 dose-dependently to reduce the expression level of H3K4mel/2/3.

Furthermore, some of the compounds of the present disclosure have alsobeen subjected to a RT-PCR experiment at the cellular level. The resultsof the inhibitory effect of some compounds on the expression levels ofdownstream Hox and Meis-1 genes at the cellular level are shown in FIG.2. As can be seen from FIG. 2, the title compound of Example 72 candown-regulate the expression levels of Hox and Meis-1 genesdose-dependently.

In addition, some of the compounds of the present disclosure have alsobeen tested for the anti-tumor activity at the animal level. A nude micexenograft tumor model of leukemia cells MV4-11 was selected for oraladministration. The results of the inhibitory effect of some compoundson the MV4-11 tumor are shown in FIG. 3. As can be seen from FIG. 3, thetitle compound of Example 72 can inhibit the tumor growthdose-dependently at the animal level.

1. An aniline-based WDR5 protein-protein interaction inhibitor,comprising a compound represented by general formula (I), apharmaceutically acceptable salt thereof, a prodrug thereof, and ahydrate or solvate thereof:

wherein X represents CH or N; Y represents C or N; R¹, R² and R³ areselected from the group consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₁-C₆ alkyl substituted with C₁-C₆ alkoxyl, nitro, halo,cyano, aldehyde group, hydroxyl, —NR⁷R⁸, or

formed by linking R² and R³, wherein R⁷ and R⁸ represent hydrogen, orC₁-C₄ alkyl, or 3- to 7-membered nitrogenous heterocycle formed bylinking R⁷ and R⁸; R⁴ represents morpholinyl, piperazinyl, 4-substitutedpiperazinyl, 4-substituted homopiperazinyl, 3-substituted piperazinyl,or 2-substituted piperazinyl, wherein a substituent is C₁-C₄ alkyl, 3-to 7-membered cycloalkyl, hydroxyalkyl, or phenyl; R⁵ represents nitro,amino, phenyl, substituted phenyl, 5- to 6-membered oxygenic ornitrogenous aromatic heterocycle, substituted 5- to 6-membered oxygenicor nitrogenous aromatic heterocycle, or —NHCOR⁹; wherein R⁹ representshydroxyl, C₁-C₆ alkoxy, phenyl, substituted phenyl, 5- to 6-memberedoxygenic or nitrogenous aromatic heterocycle, or substituted 5- to6-membered oxygenic or nitrogenous aromatic heterocycle, wherein asubstituent is C₁-C₄ alkyl, C₁-C₄ alkoxy, halo, cyano, —NHCOR¹⁰,—CONR¹¹R¹² or —COOR¹⁰, wherein R¹⁰ represents hydrogen, C₁-C₆ alkyl,C₁-C₆ amino-substituted alkyl, 3- to 7-membered cycloalkyl, 3- to7-membered nitrogenous or oxygenic heterocycle, or phenyl; and R¹¹ andR¹² represent hydrogen, C₁-C₆ alkyl, phenyl or substituted phenyl, orsubstituted or unsubstituted 3- to 7-membered nitrogenous or oxygenicheterocycle, or 3- to 7-membered nitrogenous or oxygenic heterocycleformed by linking R¹¹ and R¹²; and R⁶ represents hydrogen, halo, methyl,trifluoromethyl, amino, or substituted amino, wherein a substituent isC₁-C₄ alkyl, or allyl.
 2. The aniline-based WDR5 protein-proteininteraction inhibitor according to claim 1, wherein X represents CH orN, and Y represents C or N; wherein R¹, R² and R³ are selected from thegroup consisting of hydrogen, methyl, methoxy, nitro, halo, cyano,aldehyde group, —NR⁷R⁸, or

formed by linking R² and R³, wherein R⁷ and R⁸ represent hydrogen. orC₁-C₄ alkyl, or 3- to 5-membered nitrogenous heterocycle formed bylinking R⁷ and R⁸.
 3. The aniline-based WDR5 protein-protein interactioninhibitor according to claim 1, wherein R⁴ represents morpholinyl,piperazinyl, or 4-substituted piperazinyl, wherein a substituent ismethyl, ethyl, cyclopropyl, hydroxyethyl, or phenyl.
 4. Theaniline-based WDR5 protein-protein interaction inhibitor according toclaim 1, wherein R⁵ represents nitro, amino, —NHCOR⁹, furyl,pyrimidinyl, pyridinyl, substituted triazolyl, or substituted phenyl,wherein a substituent is a mono- or di-substituted halo, —NHCOR¹⁰,—CONR¹¹R¹², or —COOR¹⁰, wherein R⁹ represents phenyl, R¹⁰ representshydrogen, C₁-C₆ alkyl, C₁-C₆ amino-substituted alkyl, 3- to 7-memberedcycloalkyl, 3- to 7-membered nitrogenous or oxygenic heterocycle, orphenyl; and R¹¹ and R¹² represent hydrogen, C₁-C₆ alkyl, phenyl orsubstituted phenyl, substituted or unsubstituted 3- to 7-memberednitrogenous or oxygenic heterocycle, or 3- to 7-membered nitrogenous oroxygenic heterocycle formed by linking R¹¹ and R¹².
 5. The aniline-basedWDR5 protein-protein interaction inhibitor according to claim 1, whereinthe pharmaceutically acceptable salt is an inorganic acid salt or anorganic acid salt represented by general formula (I); wherein theinorganic acid salt is a hydrochloride, a hydrobromide, or a sulfate;and the organic acid salt is an acetate, a lactate, a succinate, afumarate, a maleate, a citrate, a benzoate, a methanesulfonate, or ap-toluenesulfonate.
 6. A method for preparing the general formula (I)according to claim 1, comprising following reaction steps:


7. A pharmaceutical composition, comprising the aniline-based WDR5protein-protein interaction inhibitor according to claim
 1. 8. Use ofthe aniline-based WDR5 protein-protein interaction inhibitor accordingto claim 1 in the preparation of a drug for treating an indicationrelated to a WDR5 enzyme function.
 9. The indication related to the WDR5enzyme function according to claim 8, wherein the related indication isa hematological tumor.
 10. The indication related to the WDR5 enzymefunction according to claim 9, wherein the hematological tumor is acuteleukemia.